CN101016543A - In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells - Google Patents

Abstract

The present invention relates to a high efficiency method of expressing immunoglobulin molecules in eukaryotic cells. The invention is further drawn to a method of producing immunoglobulin heavy and light chain libraries, particularly using the trimolecular recombination method, for expression in eukaryotic cells. The invention further provides methods of selecting and screening for antigen-specific immunoglobulin molecules, and antigen-specific fragments thereof. The invention also provides kits for producing, screening and selecting antigen-specific immunoglobulin molecules. Finally, the invention provides immunoglobulin molecules, and antigen-specific fragments thereof, produced by the methods provided herein.

Description

The method of screening coding for antigens specific immunoglobulin molecule or the segmental polynucleotide of its antigen-specific

The application be that November 14 calendar year 2001, application number are 01820904.1 the applying date, denomination of invention divides an application for the application for a patent for invention of " screening coding for antigens specific immunoglobulin molecule or the segmental method of its antigen-specific ".

Background of invention

Invention field

The present invention relates to a kind of method that in eukaryotic cell, efficiently expresses immunoglobulin molecules, the method in heavy chain immunoglobulin that generation is used for expressing in eukaryotic cell and light chain library, the method of the immunoglobulin (Ig) of separation and combination specific antigen, and the immunoglobulin (Ig) that produced of one of these methods.

Correlation technique

The immunoglobulin (Ig) preparation

Having specific specific antibody is just adopted by more and more different medical uses.

Specific antibodies at autoantigen is especially valuable to intravital treatment and diagnostic purpose.Utilize hybridoma technology to separate many rodent monoclonal antibodies, and these antibody are used to people's interior therapeutic and diagnostic uses.For example, one of these mouse monoclonal antibodies early stage uses is to come kill tumor or make tumor imaging as leading the target agent that (F.H.Deland and D.M.Goldenberg 1982 compile at " radionuclide-imaging " D.E.Kuhl, the 289-297 page or leaf, Pergamon, Paris; R.Levy and R.A.Miller ANN.Rev.Med.1983,34:107-116).But, use this antibody-like can cause problem in the body.The foreign immunologic sphaeroprotein can cause the anti-immunoglobulin of interference treatment and reply (R.A.Miller etc., 1983Blood62:988-995) or cause allergy or immunocomplex hypersensitization (B.Ratner, 1943, Allergy, Anaphylaxis and Immunotherapy, Williams and Wilkins, Baltimore).Accordingly, develop those in the host, for example develop in human body, itself not having immunogenic human antigen's antibody from not being that immunogenic antibody is even more important for this class application.

Separation has the task that specific antibody fragment is a difficulty to autoantigen.Animal does not produce the antibody at autoantigen usually, and this phenomenon is called tolerance.In general, carry out the generation that immunization can not cause circulating antibody with autoantigen.Therefore be difficult to cultivate antibody at autoantigen.

In the past, there were three kinds of strategies to be used to produce the immunoglobulin molecules of energy specific recognition autoantigen.In the scheme, be transplanted to people's antibody framework region by the specialization complementarity-determining region (CDR) that will comprise the antigen binding site of selecting the rodent monoclonal antibody, thereby the antibody sequence of rodent is converted into human antibody sequence (Winter etc., English Patent No.GB21886 38B (1987); Reichmann L. etc., Nature (London) 332:323-327 (1988); Foote J. and Winter G; J.Mol.Biol.224:487-499 (1992)).Be named as in antibody humanization's the method at this, three CDR rings of each heavy chain of rodent immunoglobulin (Ig) and light chain are transplanted to the same source position of four framework regions of corresponding human immunoglobulin chain.Because some framework residue also antagonist avidity has contribution, therefore structure must further be revised by other framework replacement usually, so that strengthen avidity.This may be a process that effort is expensive.

In the recent period, people have prepared the transgenic mice (MendezM.J. etc., Nat.Genet.15:146-156 (1997)) of expressing human immunoglobulin sequences.This strategy has the potentiality that acceleration is selected people's antibody, but it and antibody humanization's method have same limitation, and promptly antibody is selected from already present storehouse in the mouse, and these storehouses are that the protein of the genome encoding inhuman by mouse is formed.This may have influence on the epitope specificity at the selected antibody of specific antigen.For example, in mouse, exist the human protein of homologue to come immune mouse, may cause mainly producing the specific antibody of those different epi-positions in people and mouse with those.And these may not be best target epi-position.

One is not subjected to the alternative method of same restriction is that screening is illustrated in recombinant human antibody fragment (Vaughan T.J., the Nat.Biotechnol.14:309-314 (1996) on the phage; Barbas C.F., III, Nat.Med.1:837-839 (1995); Kay B.K. etc., " peptide and proteinic phage display ", Academic press (1996)).In the phage display method, functional immunoglobulin domains is illustrated in the surface of phage particle, and these particles have carried the polynucleotides encoding them sequence.In typical phage display method, immunoglobulin fragment, Fab for example, the stable Fv immunoglobulin domains of Fv or disulfide linkage is demonstrated with the fusion rotein form, promptly is fused on the phage surface albumen.The example that can be used to prepare the phage display method of antibody comprises (1995) (J.Immunol.Methods182:41-50 such as Brinkman U.; ); Ames, R.S. etc. (1995) are (J.Immunol.Methods184:177-186); Kettleborough, C.A. etc. (1994) are (Eur.J.Immunol.24:952-958); Persic, L. etc. (1997) are (Gene187:9-18); Burton, D.R. etc. (1994) (Advances inImmunology57:191-280); PCT/GB91/01134; WO95/15982; WO95/20401; And it is disclosed in the United States Patent (USP) 5698426,5223409,5403484,5580717,5427908,5750753,5821047,5571698,5427908,5516637,5780225,5658727 and 5733743 (the equal hereby incorporated by reference of these documents).

Because the common result of phage display method is the expression of the Fab of immunoglobulin molecules, after selecting phage, must be from phage the separating immune globulin coding region, again clone so that prepare whole antibody (comprising people's antibody) or any other purpose Fab, and in any purpose host (zooblast, insect cell, vegetable cell, yeast, bacterium), express.For example, also can use method well known in the prior art, disclosed method in for example following document, application recombinant production Fab, Fab ' and F (ab ') ₂Segmental technology: WO92/22324; (BioTechniques12 (6): 864-869 (1992) such as Mullinax R.L.; And Sawai, AJRI34:26-34 such as H. (1995); And Better, M. etc., Science240:1041-1043 (1988)) in open (the equal hereby incorporated by reference of these documents) arranged.

The immunoglobulin (Ig) library that makes up in phage can derive from the natural antibody produced cell or the individuality of specific immunity, and can comprise the new and different combinations of human immunoglobulin heavy chain and light chain in theory.Though this strategy is not subjected to the restriction of inherent antibody library, the complementary determining region (CDR) of the immunoglobulin fragment that it need be expressed can synthesize in bacterium and be correct folding.But many antigen binding domains are difficult in assembling correctly in the bacterial cell when existing with fusion rotein.In addition, protein can not carry out normal eukaryotic cell posttranslational modification.Therefore, this method has applied different selection modes for obtainable antibodies specific.

Therefore need a kind of like this or method, it can never exist in the immunoglobulin (Ig) storehouse of skewed popularity and identifies immunoglobulin molecules and antigen-specific fragment thereof, and these molecules or fragment can synthesize in eukaryotic cell, suitably glycosylation and correctly assembling.

The eukaryotic expression library.A basic tool of biology field is with poly (A) ⁺MRNA is converted into two strands (ds) cDNA, and the latter can be inserted into cloning vector and express in proper host cell then.A method commonly used comprises the construction cDNA library in many cDNA clone strategies, and this is from the poly (A) from the target organism cell ⁺MRNA deutero-cDNA clone's pond.For example, in order to separate the cDNA that expresses immunoglobulin gene, can prepare the cDNA library by pre B cell, B cell or plasmocyte.The method in construction cDNA library is known in different expression vectors (comprising filobactivirus, phage, clay and plasmid vector).In for example Sambrook etc., Molecular Cloning:ALaboratory Manual, 2 ^NdEdition, Cold Spring HarborLaboratory, publisher, Cold Spring Harbor has described some methods commonly used among the N.Y. (1990).

Many different methods that separate target gene from the cDNA library are used, have obtained success in various degree.These methods comprise, for example use dna hybrid probe, promptly have the nucleic acid fragment of crossing with the mark of the sequence of target gene dna sequence dna complementation.During to this method of cDNA clone utilization in the quilt host bacterium that transforms, those and strong colony or the plaque of hybridizing of described probe may just contain the target DNA sequence.But hybrid method does not require whether also do not detect certain specific cDNA clone is expressed.Perhaps screening method relies on the expression in the host bacterium, for example can screen colony or plaque by immunodetection, and these immunodetections detect they and bonding force at the antibody of target protein.But the detection method of expressing in the mensuration bacterial cell is often hindered, because protein may not be expressed in host bacterium efficiently, also may be expressed as wrong configuration, and perhaps it may not be processed in the eukaryotic system as and/or transport.Many above mentioned these problems have been run in the trial that the production immunoglobulin molecules is done in host bacterium.

Therefore, utilize the Mammals expression library to separate to encode the relative bacterium of the cDNA library of immunoglobulin molecules that some advantages are arranged.Immunoglobulin molecules of for example, in eucaryon host, expressing and subunit thereof should be function arranged and can carry out normal posttranslational modification.Generally the protein by cell endomembrane system transporte to cells surface should experience complete transport process.In addition, use eukaryotic system just to make and to separate polynucleotide according to eucaryotic RNA or proteinic functional expression.For example, can come the separating immune globulin molecule to the specificity of specific antigen according to them.

Except some recently by in the COS cell, express the lymphokine cDNA that is separated to (Wong, G.G. is etc., Science228:810-815 (1985); Lee, F. etc., Proc.Natl.Acad.Sci.USA83:2061-2065 (1986); Yokota, T., etc., Proc.Natl.Acad.Sci.USA83:5894-5898 (1986); Yang, Y. etc., Cell47:3-10 (1986)), from the Mammals expression library, only be separated to minority cDNA.This mainly contains two reasons: at first, the existing technology that makes up big plasmid library is difficult to grasp, and the library size is difficult to size (Huynh, T. etc. that can reach near the phage clone technology, DNA Cloning, Vol I, A Practical Approach, Glover, D.M. (volume), IRL Press, Oxford (1985), pp49-78).Secondly, existing carrier is difficult to be suitable for high level expression, has only an exception (Wong, G.G. is etc., Science228:810-815 (1985)).Therefore, in mammalian hosts, expressed in the past the proteinic identity that a kind of means of Chang Zuowei confirm the coded by said gene that those are separated to by conventional cloning process.

Poxvirus vector.Poxvirus vector is widely used as the expression vector that carries out protein and antigen presentation in eukaryotic cell.Clone and the simple and easy to do of breeding cowpox make poxvirus vector be widely used in expressing exogenous protein and as vaccine delivery carrier (Moss, B., Science252:1662-7 (1991)) in various host cells.

Big dna virus is a useful especially expression vector in the research cell processes, because they can be expressed with its natural form many different proteins in various kinds of cell system.In addition, the gene product of expressing in recombinant poxvirus demonstrates and can be processed effectively, and MHC presents the activating cytotoxic T cell with the I class.Target gene is cloned under the regulation and control that are in promotor in the plasmid usually, and wherein said promotor side is and the inessential regional homologous sequence of virus then this box to be passed through homologous recombination quiding gene group.Designed the whole series and be used for expressing, selection and the carrier that detects are realized various clones and expression strategy.But it is not the effectively means of preparation recombinant virus that homologous recombination produces under the very big situation of the DNA of complicated library or insertion at needs.An alternative strategy that makes up the recombination group has been applied to poxvirus (Merchlinsky etc., 1992, Virology 190:522-526; Pfleiderer etc., 1995, J.General Virology 76:2957-2962; Scheiflinger etc., 1992, Proc.Natl.Acad.Sci.USA 89:9977-9981), simplexvirus (Rixon etc., 1990, J.General Virology 71:2931-2939) and baculovirus (Ernst etc., 1994, Nucleic Acids Research 22:2855-2856) genome, this strategy depend on viral DNA " arm " are directly connected on the insertion fragment, and save infectious virus subsequently.

Poxvirus is a general carrier in the eukaryotic cell research, because they are easy to make up and through engineering approaches is come the high level expression exogenous protein.The extensive host range of this virus make people can be in many cell types marking protein reliably.The directed cloning strategy has been designed to expand the chimeric range of application of poxvirus virus, in this strategy by dna fragmentation directly being connected cowpox " arm " and this DNA mixture transfection is made up recombination group (Merchlinsky etc. in the cell that has infected helper virus external, 1992, Virology 190:522-526; Scheiflinger etc., 1992, Proc.Natl.Acad.Sci.USA 89:9977-9981).This method has been used to high level expression exogenous protein (Pfleiderer etc., 1995, J.General Virology 76:2957-2962), and the energy high-efficient cloning reaches the fragment (Merchlinsky etc., 1992, Virology 190:522-526) of 26kb.

Exposed vaccinia virus DNA does not have infectivity, because such virus can not be utilized cell transcription mechanism, but the protein that relies on it synthesizes viral RNA.In the past, conditioned lethal (Merchlinsky etc., 1992, Virology 190:522-526) of temperature sensitivity or nonhomologous fowlpox virus (Scheiflinger etc., 1992, Proc.Natl.Acad.Sci.USA 89:9977-9981) is used as the helper virus of packing.The ideal helper virus can assist to produce infectious virus by input DNA effectively, but can not duplicate in host cell or recombinate with vaccinia virus DNA product.Owing to these reasons, fowlpox virus is a very useful helper virus.It can enter mammalian cell and provide input vaccinia virus dna replication dna required protein.But it is not recombinated with vaccinia virus DNA, and does not produce infective fowlpox virus in mammalian cell.Therefore, can use it with higher infection multiplicity (MOI).

Usually, the exogenous protein encoding sequence is imported into the poxvirus genome group by the homologous recombination with infectious virus.In this ordinary method, the foreign DNA of prior separator well is cloned in the transferring plasmid, be positioned at after the vaccinia virus promotor, side be with poxvirus in the inessential district of virus replication homologous sequence.Described transfer vector is imported in the cell of poxvirus infection, so that this transferring plasmid and poxvirus genome group are recombinated in vivo by homologous recombination.The result of homologous recombination is that foreign DNA is transferred in the viral genome.

Though conventional homologous recombination can be used for expressing isolating foreign DNA poxvirus, this method can not help the structure in library, does not insert fragment because the viral overwhelming majority who is recovered to obtains foreign DNA.Utilize traditional homologous recombination, recombination efficiency is about 0.1% or lower.Therefore, the purposes of poxvirus vector is confined to the previous isolated DNA molecule of subclone so that carry out protein expression and vaccine development.

Set up the alternative method of utilizing direct connection carrier, so that be not suitable for carrying out to make up chimeric gene group (Merchlinsky etc., 1992, Virology 190:522-526 efficiently under the situation of homologous recombination; Scheiflinger etc., 1992, Proc.Natl.Acad.Sci.USA.89:9977-9981).In such scheme, digested from genomic DNA, be connected external with the insertion fragment, and transfection is to the cell that has infected helper virus interior (Merchlinsky etc., 1992, Virology 190:522-526; Scheiflinger etc., 1992, Proc.Natl.Acad.Sci.USA.89:9977-9981).In the scheme, genome is digested in single NotI site, and contain be useful on the DNA that selects or detect the element of mosaic gene group insert fragment be connected on the genome arm (Scheiflinger etc., 1992, Proc.Natl.Acad.Sci.USA.89:9977-9981).With this direct connection method foreign DNA is inserted into existing describe (Pfleiderer etc., 1995, J.General Virology 76:2957-2962) in the vaccinia virus gene group.

Perhaps, thereby bovine vaccine WR genome is modified generation vNotI/tk, described modification is that the NotI site in the HindIII F fragment is removed, and introduce a NotI site at the thymidine kinase gene near-end again, insert a sequence in this position like this and will destroy thymidine kinase gene, making to utilize medicament selection to come isolated chimeric gene group (Merchlinsky etc., 1992, Virology 190:522-526).

Directly connection carrier vNotI/tk makes people can clone and breed previous isolating DNA to the youthful and the elderly 26kb effectively and inserts fragment (Merchlinsky etc., 1992, Virology 190:522-526).Although big dna fragmentation is cloned in the genome effectively, this DNA inserts the coded protein of fragment and can only be expressed to be equivalent to the low-level of thymidine kinase gene, but this gene is a more weak early gene of expression ratio in the vaccinia virus.In addition, described DNA can be inserted into the NotI site with both direction, therefore may not expressed.In addition, although adopt direct-connected recombination efficiency than observed efficient height in conventional homologous recombination, the titre that obtains is lower.

Therefore, in the past came from the clonal population of complexity, to identify unknown target gene, because be not used in efficient, the cloning process that produces high titre of poxvirus without poxvirus vector.But the inventor has set up a kind of method of utilizing three molecular recombination to prepare recombinant poxvirus recently.Referring to the WO00/028016 (Zauderer) that is disclosed on May 18th, 2000, this article is the hereby incorporated by reference document herein.

Three molecular recombination are a kind of the new, efficiently and produce the method for high titre of recombinant poxvirus that are used to prepare.Adopt three molecular recombination methods in vaccinia virus, the inventor has reached at least 90% recombination efficiency, than high two orders of magnitude of titre by direct connection obtained.According to three molecular recombination methods, the poxvirus genome group is cut and produces two non-homogeneous fragments or " arm ".Prepare a transfer vector, it carries side is allos inset DNA with two poxvirus arm homologous zones.Arm and transfer vector are delivered to the acceptor host cell, make these three dna moleculars to recombinate in vivo.The result of reorganization has produced the poxvirus genome group unit molecule that comprises two poxvirus arms and described insertion DNA.

Summary of the invention

Corresponding one aspect of the present invention provides a kind of like this method, this method can be from the polynucleotide library of eukaryotic cell, expressing identification code antigen specific immune globulin molecule or the segmental polynucleotide of its antigen-specific.

Provide the method for a kind of identification code immunoglobulin molecules or its segmental polynucleotide simultaneously, described molecule or its fragment have the effector function that changes.

A kind of method of utilizing virus vector to make up the polynucleotide library of coding immunoglobulin (Ig) subunit polypeptide in eukaryotic cell also is provided, and wherein said library makes up by three molecular recombination methods.

The method of identifying antigen expressed specific immunoglobulin molecule in its surface or the segmental host cell of its antigen-specific is provided in addition, described evaluation be by to the signal transmission of the necrocytosis of antigen inductive, antigen induction or antigen-specific in conjunction with selecting and/or screen realization.

The solubility immunoglobulin molecules or the segmental screening method of its antigen-specific of being expressed by eukaryotic host cell also are provided, the polynucleotide library of the immunoglobulin,exocrine molecule of described host cell expression coding solubility, screening are to carry out by the antigen combination or by the antigen or the organism specific function that detect described immunoglobulin molecules.

The accompanying drawing summary

Fig. 1: the apoptosis by antigen induction is selected human antibodies specific.

Fig. 2 A: the antigen cross-linking of preparation response surface immunoglobulin (Ig) and directly or indirectly experience the host cell of necrocytosis.

Fig. 2 B: the confirmation of the CH33 host cell of modification, this cell are designed to when the antigen cross-linking of surface immumoglobulin cracking of CTL-inductive or necrocytosis to take place.

The structure of Fig. 3: pVHE.

The structure of Fig. 4: pVKE and pVLE.

Fig. 5: adhere to by the antigen dependency and to select specific human antibodies.

Fig. 6: the synoptic diagram of three molecular recombination methods.

The nucleotide sequence of Fig. 7: p7.5/tk and pEL/tk promotor.The figure illustrates the nucleotide sequence that described promotor and thymidine kinase gene begin to locate and (for V7.5/tk, be SEQ ID NO:140; For VEL/tk, be SEQ ID NO:142), and the aminoacid sequence that comprises atg start codon and part opening code-reading frame accordingly, distinguish called after SEQID NO:141 and SEQ ID NO:143 herein.

The structure of Fig. 8: pVHEs.

Fig. 9: the weakening of the cytopathic effect of poxvirus mediation.

Figure 10: the structure of scFv expression vector.

The structure of Figure 11: pVHE-X-G1.

A modification (SEQID NO:1) of the nucleotide sequence of Figure 12 A:p7.5/tk bovine vaccine transferring plasmid.One as described herein by p7.5/tk bovine vaccine transferring plasmid deutero-novel vector p7.5/ATGO/tk (SEQ ID NO:2).

Figure 12 B: as described herein by p7.5/tk bovine vaccine transferring plasmid deutero-novel vector p7.5/ATG1/tk (SEQ ID NO:3).

Figure 12 C: as described herein by p7.5/tk bovine vaccine transferring plasmid deutero-novel vector p7.5/ATG2/tk (SEQ ID NO:4).

Figure 12 D: as described herein by p7.5/tk bovine vaccine transferring plasmid deutero-novel vector p7.5/ATG3/tk (SEQ ID NO:5).

Figure 13: the structure of IgM-Fas fusion product.

Figure 14: Ig α and the Ig β expression in transfected COS7 and HeLaS3 clone.Total RNA separates from (A) COS7-Ig α β-1, (B) COS7-Ig α β-2, (C) HeLaS3-Ig α β-1, (D) human B cell of EBV-conversion, be transcribed into cDNA in the situation subinverse that is with or without reversed transcriptive enzyme, use ig α 5 '/ig α 3 ' and ig β 5 '/ig β 3 ' primer sets to carry out pcr amplification then.On 0.8% sepharose, analyze the PCR product then.Should be noted that human B cell all demonstrates other montage mode to Ig α and Ig β.Referring to for example, Hashimoto, S. etc., Mol.Immunol.32:651 (1995).

DESCRIPTION OF THE PREFERRED

The present invention relates generally in eukaryotic system to identify and/or produces the method for functional, antigen specific immune globulin molecule or its antigen-specific fragment (being Fab).In addition, the present invention relates to the method for from coding for antigens specific immunoglobulin molecule or the compound expression library of the segmental polynucleotide of its antigen-specific these immunoglobulin molecules of identification code or segmental polynucleotide, wherein said library makes up in eukaryotic host cell and expresses.Other embodiments comprise by the isolating antigen specific immune globulin molecule of any method for preparing or its antigen-specific fragment, and the test kit that can prepare the isolating immunoglobulin (Ig) of this class.

Particularly preferred aspect of the present invention is to utilize the poxvirus vector that makes up by three molecular recombination to make up the complex immunity globulin library in eukaryotic host cell.In based on the carrier of poxvirus, make up the ability in compound cDNA library, and according to the signal transmission of the necrocytosis of antigen induction, antigen induction or antigen-specific in conjunction with the ability of selecting and/or screening specific recombinant chou, can be used as and in eukaryotic cell, identify immunoglobulin (Ig), especially have a basis of many very clear and definite specific human normal immunoglobulins.It can overcome the skewed popularity that causes when the antagonist storehouse is selected in rodent, the perhaps limitation of synthesizing and assembling in phage or bacterium.

Should be mentioned that term " " or " a kind of ", refer to " one or more (kind) "; For example, " immunoglobulin molecules " is interpreted as representing one or more immunoglobulin molecules.Therefore, term " (kind) ", " one or more (kind) " and " at least one (kind) " can exchange in the text.

Term " eukaryote (eukaryote) " or " eukaryote (eukaryoticorganism) " are meant all biologies of containing in animal, plant and the Protista, comprise protozoon, fungi, yeast, green alga, one-celled plants, metaphyte and all animals comprise vertebra and invertebrates.This term does not comprise bacterium and virus." eukaryotic cell " is meant and comprises single " eukaryotic cell " and a plurality of " eukaryotic cells ", and contain from Eukaryotic cell.

Term " vertebrates " is meant contains single " vertebrates " and a plurality of " vertebratess ", comprises Mammals and birds, and fish, reptiles and batrachians.

Term " Mammals " is meant contains single " Mammals " and a plurality of " Mammalss ", includes but not limited to the people; Primates is such as ape, monkey, orangutan and chimpanzee; Canidae is such as dog and wolf; Cat family is such as cat, lion and tiger; Equine is such as horse, donkey and zebra; Edible animal is such as ox, pig and sheep; Ungulate is such as deer and giraffe; Rodents is such as mouse, rat, hamster and cavy; And bear.Preferably, described Mammals is the people.

Term " tissue culture " or " cell cultures " or " cultivation " be meant can preserve cellularstructure, cell function, further under differentiation or all conditions of three kinds, plant or animal tissues or cells in vitro are kept or are grown." former generation histocyte " is those cells of directly taking from tissue, promptly carries out the similar cell mass of identical function in organism.With this class histocyte of proteolytic ferment trypsin treatment, they can be dissociated into and grow or to keep cyto-architectural single former generation histocyte when being seeded on the culture plate.The cell culture that is obtained by primary cell propagation in tissue culture is called " secondary cell culture ".The death then several times that most secondary cell divisions are limited.But the minority secondary cell can pass through this " critical days ", thereby they can form " clone " that continues by infinite multiplication afterwards.The liquid nutrient medium that cell is cultivated therein is called " substratum " or " developing medium " in the text.In cell cultivation process molecules of interest for example the substratum that is secreted into wherein of immunoglobulin molecules be called " conditioned medium " in the text.

Term " polynucleotide " refers to be present in any one or more nucleic acid segment in nucleic acid or the construct, perhaps nucleic acid molecule, for example DNA or RNA fragment.The polynucleotide of subunit polypeptide " coding immunoglobulin (Ig) " refers to comprise the polynucleotide of the coding region of described polypeptide.In addition, the polynucleotide controlling element of may encoding, such as promotor or transcription terminator, perhaps may coded polypeptide or proteinic particular components, such as secreting signal peptide or functional structure territory.

With in the text, term " evaluation " is meant a kind of like this method, and this method can be with molecules of interest, and for example the polynucleotide of coding with immunoglobulin molecules of required specificity or function distinguish from the colony of this quasi-molecule or library.Authentication method comprises " selection " and " screening ".With in the text, " selection " method is those methods that can directly isolate molecules of interest from the library.For example, in the system of selection of describing in the literary composition, by a hydrolysis, the host cell that comprises polynucleotide of interest discharges from matrix, and the cell that comprises other polynucleotide in the library still adheres on it, thereby they are directly separated.With in the text, " screening " method is that those wherein carry out a kind of method that can detect the analysis of molecules of interest to the pond that comprises molecules of interest.A pond that will detect described molecule then is divided into diminishing pond by similar analysis, up to the pond that obtains highly to be rich in molecules of interest.For example, in the text in the sieve method of Miao Shuing, by expressing reporter molecules, its antigen bonding force is analyzed in the host cell pond in the polynucleotide library that comprises the immunoglobulin molecules of encoding.

Immunoglobulin (Ig).With in the text, " immunoglobulin molecules " is defined as complete bimolecular immunoglobulin (Ig), promptly comprises four " subunit polypeptides " usually, i.e. two identical heavy chains and two identical light chains.In some cases, for example, derive from the camelid species or on camelid immunoglobulin (Ig) basis the immunoglobulin molecules of through engineering approaches, complete immunoglobulin molecules may only be made of heavy chain, does not have light chain.Referring to for example Hamers-Casterman etc., Nature363:446-448 (1993).Therefore " immunoglobulin (Ig) subunit polypeptide " is meant single heavy chain polypeptide or single light chain polypeptide.Immunoglobulin molecules also can be called " antibody ", and these two terms can exchange use in the text." isolating immunoglobulin (Ig) " is meant an immunoglobulin molecules, perhaps two or more immunoglobulin molecules, and they break away from the environment of protein and other materials basically, and can be in conjunction with specific antigen.

The heavy chain of " class " of decision immunoglobulin molecules is bigger in two subunit polypeptides, and it comprises variable region and constant region." heavy chain " is meant excretory total length heavy chain form, the form that promptly from cell, discharges, and perhaps film promptly comprises membrane spaning domain and born of the same parents' intracellular domain in conjunction with the heavy chain form.Striding film can be the relevant structural domain of naturally occurring and a certain heavy chain with born of the same parents' intracellular domain, promptly be found in the structural domain in the memory B cell, perhaps it may be that allos is striden film and born of the same parents' intracellular domain, for example from different types of immunoglobulin (Ig) or from heterologous polypeptide, i.e. many skins of NIg.Be apparent that, preferably use cytolemma mating type immunoglobulin molecules to implement some aspect of the present invention, and other aspects preferably use secretory immunoglobulin molecule (being that those shortages are striden film and born of the same parents' intracellular domain) to implement.Immunoglobulin (Ig) " kind " is meant in the host big group of immunoglobulin (Ig) carrying out difference in functionality.For example human normal immunoglobulin can fall into 5 types, i.e. IgG (containing gamma heavy chain), IgM (containing the μ heavy chain), IgA (containing the α heavy chain), IgE (containing the ε heavy chain) and IgD (containing the δ heavy chain).The immunoglobulin (Ig) of some kind is further divided into " subclass " again.For example, four kinds of different IgG subclass are arranged in human body, promptly comprise IgG1, IgG2, IgG3 and the IgG4 of γ-1, γ-2, γ-3 and γ-4 heavy chain respectively, and two different IgA subclass, promptly comprise the IgA-1 and the IgA-2 of α-1 and α-2 heavy chains respectively.The class and the subclass name that should be mentioned that immunoglobulin (Ig) are different between animal species, and some animal species may comprise the immunoglobulin (Ig) of other kind.For example, birds also produce the IgY that is included in the yolk.

" light chain " is meant the less immunoglobulin (Ig) subunit that connects together with heavy chain amino terminal zone.The same with heavy chain, light chain comprises variable region and constant region.It is κ and λ that two kinds of different light chains are arranged, and a pair of such light chain can combine with any a pair of heavy chain, forms immunoglobulin molecules.

Each comprises constant region and variable region the immunoglobulin (Ig) subunit polypeptide.In most species, variable region of heavy chain or V _HStructural domain, and variable region of light chain or V _LStructural domain is combined together to form " complementary determining region " or CDR, and it is the part of specific recognition epitope in the immunoglobulin molecules.But in camel (camelid) species, be called as V _HThe variable region of heavy chain of H constitutes whole C DR.Camel V _HH variable region and from the variable region (V of conventional antibody _H) the key distinction comprise (a) V _HThe light chain surface in contact compare V _HRespective regions among the H has more hydrophobic amino acids, (b) V _HThe CDR3 of H is longer, and (c) V _HOften there is disulfide linkage among the H between CDR1 and the CDR3.Each complete immunoglobulin molecules comprises two identical CDR.Animal is at the antibody produced cell differentiation phase, by a series of kinds be the rearrangement of DNA section, cause forming the gene in certain particular variable district of coding, thus the huge pond of can generation relevant variable region with heavy chain and constant region of light chain.Other variations of heavy chain and variable region of light chain can realize by the somatic mutation in the noble cells.The those of ordinary skill that the interior formation of the structure of immunoglobulin molecules and body is field of immunology is known.About the simple and clear summary of the multifarious generation of immunoglobulin (Ig) as seen for example, Harlow and Lane, Antibodies, A Laboratory ManualCold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1988) (hereinafter referred to as " Harlow "); And Roitt etc., Immunology Gower Medical Publishing, Ltd., London (1985) (hereinafter referred to as Roitt ").Harlow and Roitt be hereby incorporated by reference herein.

Immunoglobulin (Ig) also has some in addition by combine the effector function that mediates with effector molecule.For example, the C1 component of complement and immunoglobulin (Ig) bound energy activating complement system.The opsonization and the cracking of the activation pair cell pathogenic agent of complement are very important.The activation of complement can also stimulate inflammatory reaction, also might participate in the autoimmunization allergy.In addition, by the Fc zone, the Fc acceptor site on the antibody Fc zone combines with Fc acceptor (FcR) on the cell, thereby immunoglobulin (Ig) is attached on the cell.Many Fc acceptors that are specific to inhomogeneity antibody are arranged, include but not limited to, IgG (γ acceptor), IgE (η acceptor), IgA (α acceptor) and IgM (μ acceptor).The combination of the Fc acceptor on antibody and the cell surface excited many will with different biological responses, comprise the particulate of coated antibody is engulfed and destroyed, remove immunocomplex, killer cell is to the cracking (cytotoxicity that is called the antibody dependent cellular mediation of the target cell of coated antibody, or ADCC), the release of mediator of inflammation, placental transport and regulation and control that immunoglobulin (Ig) produces.

Immunoglobulin (Ig) of the present invention can comprise birds from any animal-origin, fish and Mammals.Preferably, described antibody sources is in the people, mouse, dog, cat, rabbit, goat, cavy, camel, vigone (llama), horse or chicken.Of the present invention one preferred aspect, identified and the interactional immunoglobulin (Ig) in " self " antigen-specific ground, for example specific combination human antigen's human normal immunoglobulin.

" the antigen-specific fragment " of the immunoglobulin molecules that uses in the literary composition is any fragment or the variant that has kept antigen binding capacity in the immunoglobulin molecules.The antigen-specific fragment includes, but are not limited to Fab, Fab ' and F (ab ') ₂Fd, strand Fvs (scFvs), strand immunoglobulin (Ig) (for example, heavy chain or its part and light chain or its meromixis immunoglobulin (Ig) together), the Fvs (sdFvs) that disulfide linkage connects, disome (diabodies), trisome (triabodies), limbs (tetrabodies), scFvs microbody (minibodies), Fab microbody (minibodies) and dimer scFvs and any other comprise V _LAnd V _HStructural domain and these structural domains are in the fragment that can form the conformation of special CDR.The antigen-specific fragment also may comprise the V that derives from camel id antibody _HThe H structural domain.V wherein _HThereby H can be comprised CDR from other species (for example from people's antibody) by through engineering approaches.Perhaps, can be with the heavy chain V in people source _HIt is similar to strand camelid CDR that the fragment through engineering approaches makes it, and this process is called " camel sourceization (camelization) ".Referring to for example, Davies J. and Riechmann, L., FEBS Letters 339:285-290 (1994) and Riechmann, L. and Muyldermans, S., J.Immunol.Meth.231:25-38 (1999), the equal herein hereby incorporated by reference of these two pieces of articles.

Antigen specific immune sphaeroprotein fragment (comprising the strand immunoglobulin (Ig)), may only comprise the variable region or with the combination in whole or in part of following material: heavy chain constant domain or its part, the CH1 on the heavy chain for example, CH2, CH3 strides film and/or tenuigenin structural domain; And constant region of light chain, for example C κ or C λ structural domain, or the part on the light chain.Comprise simultaneously in the present invention be variable region and CH1, CH2, CH3, C _κ, C _λ, stride any array configuration of film and tenuigenin structural domain.

Known in this field, Fv comprises VH structural domain and VL structural domain, and Fab comprises VH and the L chain that is connected on the CH1, and Fab microbody (minibody) comprises the fusion of CH3 structural domain and Fab etc.

Known in this field, it is the VH that the peptide linker of 15-20 residue is connected to VL that scFv comprises by general length, disome (diabodi es) comprises the scFv of the peptide linker that has 5 residues of about length, trisome (triabodies) comprises the scFv that is not with peptide linker, limbs (tetrabodies) comprise and have length is the scFv of the peptide linker of 1 residue, scFv microbody (minibody) comprises the scFv that has merged the CH3 structural domain, dimer scFv comprises two scFv syzygys (summary is seen Chames and Baty, FEMS Microbiol.Letts.189:1-8 (2000)) of utilizing another peptide linker to be cascaded.Preferably, described antigen specific immune sphaeroprotein fragment comprises two kinds of antigen binding domainss, i.e. V _HAnd V _LOther immunoglobulin fragments are well known in the art, all have open in these reference of describing in such as literary composition.

In certain embodiments, the present invention relates to identify the method for (promptly selecting or screening) polynucleotide, independent or the common coding for antigens specific immunoglobulin of described polynucleotide molecule, its antigen-specific fragment or have the immunoglobulin molecules or the fragment of specific antigens correlation function.In relevant embodiment, the present invention relates to the coded isolating immunoglobulin molecules of polynucleotide that identifies by these methods.

Preferable methods comprises screening of two steps and/or the process of selecting.In first step, the library of the polynucleotide by the first immunoglobulin (Ig) subunit of will encoding imports eukaryotic host cell colony, and express described subunit with one or more second immunoglobulin (Ig) subunit, thereby by the polynucleotide that identify the coding first immunoglobulin (Ig) subunit (being heavy chain or light chain) in this library, here this second immunoglobulin (Ig) subunit is different with the first immunoglobulin (Ig) subunit, if promptly the first immunoglobulin (Ig) subunit is a heavy chain polypeptide, then the second immunoglobulin (Ig) subunit is exactly a light chain polypeptide.

In case from the library, be separated to one or more polynucleotide of one or more first immunoglobulin (Ig) subunit of coding in a first step, just in second step, identify the second immunoglobulin (Ig) subunit.The isolating polynucleotide of the isolating first immunoglobulin (Ig) subunit of coding are transferred to host cell and expressed, in the encode polynucleotide library of the second immunoglobulin (Ig) subunit of described cell expressing, so make the polynucleotide that can identify the coding second immunoglobulin (Ig) subunit, this second immunoglobulin (Ig) subunit has just formed functional immunity globulin molecule or its fragment that can discern specific antigen and/or carry out exceptional function when combining with the first immunoglobulin (Ig) subunit that identifies in first step.

(be single-chain fragment or comprise V when immunoglobulin fragment only is made of a peptide species _HThe fragment of H structural domain), therefore be during by a kind of polynucleotide encoding, preferable methods comprises step screening and/or the process of selecting.By the library being imported host cell (such as eukaryotic cell), and the polynucleotide of the immunoglobulin fragment of from these host cells, encoding in this library of recovery, thereby from described library, identify the polynucleotide of coding single-chain fragment, wherein said single-chain fragment comprises variable region of heavy chain and variable region of light chain, perhaps comprises V _HThe H district.

In certain embodiments, contact with antigen by the host cell with expressing immunoglobulin molecule on the surface and to identify specific immunoglobulin molecules, this just makes can select and/or screen ABC with the many different methods that describe below.In other embodiments, by the required function characteristics (for example virus neutralization) of the concentrated immunoglobulin molecules of analysis condition substratum, can identify the immunoglobulin,exocrine molecule of required solubility.

Be combined in the situation of host cell surface at immunoglobulin molecules, first step comprises that the first polynucleotide library with the multiple first immunoglobulin (Ig) subunit polypeptide of coding, the transcription regulatory region that has been operably connected imports to the host cell group that can express described immunoglobulin molecules; In identical host cell, import the coding second immunoglobulin (Ig) subunit polypeptide group, operably connected the second polynucleotide library of transcription regulatory region; In described host cell surface expressing immunoglobulin molecule or its antigen-specific fragment; Host cell is contacted with antigen, and from those host cells of antigen bonded retrieve polynucleotide from first library.

Be secreted into fully in the situation of cell culture medium at immunoglobulin molecules, first step comprises that the first polynucleotide library that with the coding first immunoglobulin (Ig) subunit polypeptide group, has operably connected transcription regulatory region imports among the host cell group that can express described immunoglobulin molecules; In identical host cell, import the coding second immunoglobulin (Ig) subunit polypeptide group, operably connected the second polynucleotide library of transcription regulatory region; Make immunoglobulin molecules or its antigen-specific fragment secreting, expressing justacrine in cell culture medium; Whether has the relevant antibody function of required antigen in the sample of analysis condition substratum; And in being grown in host cell the conditioned medium of observing required function, those retrieve polynucleotide from first library.

In the text, " library " is the representative classes of polynucleotide, be such a group polynucleotide, they are because of for example interrelated from same animal species, types of organization, organ or cell type, and wherein said library is total, and to comprise given polynucleotide at least two in belonging to not of the same race.The preferred polynucleotide library comprises at least 10,100,10 in given polynucleotide belong to ³, 10 ⁴, 10 ⁵, 10 ⁶, 10 ⁷, 10 ⁸Or 10 ⁹Individual not of the same race.More particularly, library of the present invention coding a plurality of certain immunoglobulin (Ig) subunit polypeptides, i.e. heavy chain subunit polypeptide or light chain subunit polypeptide.In this article, " library " of the present invention includes the polynucleotide of predicable, and this genus is the polynucleotide of the immunoglobulin (Ig) subunit polypeptide of coding ad hoc type and class.For example, the library people μ that may encode, γ-1, γ-2, γ-3, γ-4, α-1, α-2, ε or δ heavy chain, perhaps encode people κ or lambda light chain.Identical heavy chain or constant region of light chain though the member in any one library of the present invention encodes, the library totally comprises at least 2, and preferably at least 10,100,10 ³, 10 ⁴, 10 ⁵, 10 ⁶, 10 ⁷, 10 ⁸Or 10 ⁹Individual different variable region promptly has the relevant a plurality of variable regions of constant region together.

In other embodiments, library a plurality of immunoglobulin (Ig) single-chain fragments of encoding, described fragment comprises the variable region, such as variable region of light chain or variable region of heavy chain, has preferably both comprised variable region of light chain and has also comprised variable region of heavy chain.Choose wantonly, this class library comprises a certain type of coding and the many skins of immunoglobulin (Ig) subunit of class or the polynucleotide of its structural domain.

One aspect of the present invention is contained the method in the polynucleotide library of preparation coding immunoglobulin (Ig) subunit.In addition, the immunoglobulin (Ig) subunit library that makes up according to the method for describing in the literary composition is contained in the present invention in carrier for expression of eukaryon.Preferred this class library is at the eucaryon virus vector, more preferably prepare in the poxvirus vector.Such method and library have been described in the literary composition.

" recipient cell " or " host cell " or " cell " are meant cell or the cell mass that has wherein imported polynucleotide of the present invention library.Host cell of the present invention is eukaryotic cell or clone preferably, preferably plant, animal, vertebrates, Mammals, rodent, mouse, primate or people's cell or clone." host cell group " is meant a group culturing cell that " library " of the present invention can be imported into wherein and express.Any host cell that support is structured in the expression in the given library in the given carrier is all included.Suitable and preferred host cell are disclosed in the literary composition.In addition, disclose preferably with concrete carrier in the literary composition or specifically selected and/or particular host cell that screening scheme uses.Though the preferred host cell group is a monoculture, promptly each cell in the colony is identical cell type, also can consider the cytomixis culture.Host cell of the present invention can adhere to, and promptly is attached to host cell grown on the solid substrate, and perhaps, host cell can suspension growth.Host cell can be the cell that derives from primary tumo(u)r, derives from the cell of metastatic tumor, primary cell, the cell of forfeiture contact inhibition, the primary cell that transforms, the primary cell of immortalization may experience the cell of apoptosis and by these cell-derived clone.

As mentioned above, identify that the preferred method of immunoglobulin molecules comprises first polynucleotide library importing host cell group, and import the second polynucleotide library to identical host cell group.Described first and second libraries are complementary, if i.e. first library coding heavy chain immunoglobulin, second library light chain immunoglobulin of then encoding, thus can in the host cell group, be assembled into immunoglobulin molecules or its antigen-specific fragment.As mentioned above equally, identify that the another kind of method of immunoglobulin (Ig) or immunoglobulin fragment comprises that the single polynucleotide library with the coding single-chain fragment imports the host cell group.Therefore to the description in polynucleotide library, the composition of polynucleotide in the library, and contained the polynucleotide that constitute first library and constituted the polynucleotide in second library by the polypeptide of these polynucleotide encodings, and their encoded polypeptides.Can in any suitable carriers, make up these libraries, and two kinds of libraries can but and nonessential being structured in the identical carrier.Suitable and the preferred carrier that is used for first and second libraries is hereinafter disclosed.

Be included in polynucleotide in the library of the present invention by " operably being connected " with transcription regulatory region thus coding immunoglobulin (Ig) subunit polypeptide.One or more nucleic acid molecule in given polynucleotide is in functional relationship, and they " operably connect ".This relation may reside between the coding region and regulating and controlling sequence of polypeptide, and their ways of connecting make that when being attached on the regulating and controlling sequence, the coding region will be expressed when suitable molecule (for example, transcription activating protein, polysaccharase etc.)." transcription regulatory region " includes, but are not limited to promotor, enhanser, and operon and transcription termination signal, and it exists to instruct transcribing of it with polynucleotide.For example, if promotor can influence the transcribing of nucleic acid molecule of coding immunoglobulin (Ig) subunit polypeptide, then this promotor operably links together with described nucleic acid molecule.Usually, " operably connect " is meant in the polynucleotide that dna sequence dna adjoins or links together closely.But some transcription regulatory region, for example enhanser not necessarily adjoins.

" regulating and controlling sequence " or " control region " is meant and expresses the necessary dna sequence dna of encoding sequence that can handle connection in the specific host biology.Be applicable to procaryotic regulating and controlling sequence, for example comprise promotor, optional operon sequence, and ribosome bind site.The known genuine karyocyte can utilize promotor, polyadenylation signal and enhanser.

Various transcription regulatory regions are known in those skilled in the art.Preferred transcription regulatory region comprises what those can play a role in vertebrate cells, such as comprising, but be not limited to from poxvirus, adenovirus, simplexvirus, human cytomegalic inclusion disease virus (early promoter in preferred for example, and preferably connected intron A), simian virus 40 (preferred early promoter), retrovirus (such as Rous sarcoma virus) and picornavirus (particularly internal ribosome entry site or IRES, strengthen the subarea, be called the CITE sequence in the literary composition again) promotor and enhancer sequence.Other preferred transcription regulatory regions comprise that those derive from mammalian genes, and such as the control region of Actin muscle, heat shock protein(HSP) and Trobest, and other energy controlling genes are in the sequence of expression in eukaryotic cells.Suitable in addition transcription regulatory region comprises tissue-specific promoter and enhanser and inducible promoter (for example can be by tsiklomitsin inductive promotor, and temperature sensitivity promotor).Particularly preferably be the promotor that can play a role in by the tenuigenin of the cell of poxvirus infection, this will go through hereinafter.

In some preferred embodiment, each " immunoglobulin (Ig) subunit polypeptide " (i.e. " first immunoglobulin (Ig) subunit polypeptide " or " the second immunoglobulin (Ig) subunit polypeptide ") comprises first constant region for immunoglobulin that (i) is selected from CH (the film combining form of CH or complete secreted form) and constant region of light chain, (ii) corresponding immune globulin variable region with first constant region, if promptly described constant region for immunoglobulin is a CH, then immune globulin variable region preferably comprises V _HThe district, and if constant region for immunoglobulin is a variable region of light chain, then the preferred immunoglobulin constant region comprises V _LDistrict, and (iii) can guide the immunoglobulin (Ig) subunit polypeptide to pass the signal peptide that endoplasmic reticulum and host cell plasma membrane transport, wherein said immunoglobulin (Ig) subunit polypeptide be film in conjunction with or excretory heavy chain fully, perhaps be connected with the light chain of heavy chain.Therefore, by mutually combining of two identical heavy chains and two identical light chains, can form surface immumoglobulin molecule or complete excretory immunoglobulin molecules.

In some preferred embodiment about immunoglobulin fragment, single-chain fragment comprises immune globulin variable region, and it is selected from variable region of heavy chain and variable region of light chain, preferably includes two kinds of variable regions.If described immunoglobulin fragment not only comprises variable region of heavy chain but also comprise variable region of light chain, they can be (being the joint that they do not have peptide or other kinds) that directly connects together, and perhaps connect by other means.As mentioned below, if they otherwise connect, they can be direct-connected, and perhaps the disulfide linkage by forming in the expression process perhaps connects by peptide linker.Accordingly, the associating by variable region of heavy chain and variable region of light chain has just formed CDR.

Variable region of heavy chain and variable region of light chain in single-chain fragment can be interrelated, and perhaps the variable region of heavy chain of a single-chain fragment can be related with the variable region of light chain of another one single-chain fragment, and vice versa, and this depends on the type of joint.In one embodiment, described single-chain fragment can also comprise constant region, and it is selected from the group of being made up of CH or its structural domain and constant region of light chain or its structural domain.Two single-chain fragments can be interrelated by their constant region.

As mentioned above, in certain embodiments, the variable region of light chain of the described single-chain fragment of encoding and the polynucleotide of the variable region of heavy chain joint of also encoding.Described single-chain fragment may comprise and has V _H-joint-V _LPerhaps V _L-joint-V _HThe single polypeptide of sequence.In some embodiments, selected joint can combine the heavy chain of single-chain fragment and light chain with suitable conformation direction.Referring to, Huston for example, J.S. etc., Methods in Enzym.203:46-121 (1991).Therefore in these embodiments, joint should cross over it with the merging point of variable region between 3.5nm apart from and non-warping natural Fv conformation.In these embodiments, the amino-acid residue that constitutes joint can be crossed over this distance, and 5 amino acid or longer should be arranged.The single-chain fragment that has 5 amino acid joints can exist by monomeric form, but mainly is dimeric forms.Preferably, described joint grows up about 10 or at least 15 residues at least.In other embodiments, the selection of butt junction length is wanted can promote to form the scFv tetramer (limbs), and it is 1 amino acid long.In some embodiments, the variable region is connected directly (being that single-chain fragment does not contain peptide linker) so that promote to form scFv tripolymer (trisome).These variations are (referring to for example, Chames and Baty, FEMSMicrobiol.Letts.189:1-8 (2000)) well known in the art.Joint should not grown and cause binding site is taken place by three-dimensional the interference.Therefore, preferred joint about 25 residues or shorter of growing up.

The amino acid of preferred selected peptide linker makes that joint is hydrophilic, and it can not be embedded in the antibody like this.Joint (Gly-Gly-Gly-Gly-Ser) ₃(SEQ ID NO:6) is a preferred joint that is widely used in many antibody, because it can provide sufficient flexibility.Other joints comprise Glu Ser Gly Arg Ser Gly Gly Gly Gly Ser Gly Gly Gly GlySer (SEQ ID NO:7), Glu Gly Lys Ser Ser Gly Ser Gly Ser GluSer Lys Ser Thr (SEQ ID NO:8), Glu Gly Lys Ser Ser Gly SerGly Ser Glu Ser Lys Ser Thr Gln (SEQ ID NO:9), Glu Gly LysSer Ser Gly Ser Gly Ser Glu Ser Lys Val Asp (SEQ IDNO:10), Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly LysGly (SEQ ID NO:11), Lys Glu Ser Gly Ser Val Ser Ser Glu GlnLeu Ala Gln Phe Arg Ser Leu Asp (SEQ ID NO:12), and Glu SerGly Ser Val Ser Ser Glu Glu Leu Ala Phe Arg Ser Leu Asp (SEQID NO:13).Perhaps, butt junction is carried out mutagenesis such as (Gly-Gly-Gly-Gly-Ser) 3 (SEQ IDNO:6), perhaps with the amino acid randomization in the joint, utilize Vector for Phage Display or method of the present invention, it is the highest or phenotype had the greatest impact that screening or select has avidity in the antibody of different joints, can certainly use any sequence.Comprise the fragment of above-mentioned joint than the example of short circuit head, the example of lengthening joint comprises the array configuration of above-mentioned joint, the segmental array configuration of above-mentioned joint, and the array configuration of above-mentioned joint and linker fragment.

Equally preferably as variant or segmental those immunoglobulin (Ig) subunit polypeptides of immunoglobulin (Ig) subunit polypeptide described above.In the variant of any Fab that can produce immunoglobulin molecules or fragment all are encompassed in.This class variant may by for example with natural the related of film district of striding, receptor-ligand binding is perhaps striden the fusion in film district with allos and attached to host cell surface, perhaps may be secreted in the cell culture medium.The example of the Fab of immunoglobulin molecules has been described in the literary composition.

Comprise in the embodiment of heavy chain polypeptide at those immunoglobulin (Ig) subunit polypeptides, all included from any heavy chain immunoglobulin of any animal species.Described suitable in the literary composition and the preferred immunoglobulins heavy chain.Described heavy chain immunoglobulin comprises that from vertebrates such as bird (particularly chicken), fish and mammiferous heavy chain, wherein mammalian immune sphaeroprotein heavy chain is preferred.The example of heavy chain immunoglobulin comprises the people, mouse, dog, cat, horse, goat, rat, sheep, ox, pig, cavy, camel, vigone and hamster heavy chain immunoglobulin.In these, the human immunoglobulin heavy chain is especially preferred.What it is also conceivable that is the heterozygosis heavy chain immunoglobulin, and it comprises the heavy chain part from one or more species, such as mouse/people's heterozygosis heavy chain immunoglobulin, the perhaps human immunoglobulin heavy chain of " camel sourceization ".For the human immunoglobulin heavy chain, preferred heavy chain immunoglobulin of the present invention is selected from following group: μ heavy chain (being the heavy chain of IgM immunoglobulin (Ig)), γ-1 heavy chain (being the heavy chain of IgG1 immunoglobulin (Ig)), γ-2 heavy chain (being the heavy chain of IgG2 immunoglobulin (Ig)), γ-3 heavy chain (being the heavy chain of IgG3 immunoglobulin (Ig)), γ-4 heavy chain (being the heavy chain of IgG4 immunoglobulin (Ig)), α-1 heavy chain (being the heavy chain of IgA1 immunoglobulin (Ig)), α-2 heavy chain (being the heavy chain of IgA2 immunoglobulin (Ig)), ε heavy chain (being the heavy chain of IgE immunoglobulin (Ig)), and δ heavy chain (being the heavy chain of IgD immunoglobulin (Ig)).In certain embodiments, the preferred immunoglobulins heavy chain comprises the people μ of film combining form, γ-1, γ-2, γ-3, γ-4, α-1, α-2, ε and δ heavy chain.Particularly preferably be the people μ heavy chain of film combining form.

The immunoglobulin (Ig) of film combining form normally is anchored at cell surface by membrane spaning domain, wherein stride the film district through the alternative transcription termination of heavy chain messenger RNA(mRNA) and montage and constitute the part of heavy chain polypeptide.Referring to for example Roitt the 9th and 10 pages." membrane spaning domain ", " striding the film district " or the relational language that can exchange are meant a part that is anchored at the heavy chain polypeptide on the cytolemma.Typical membrane spaning domain comprises hydrophobic amino acid, and this will discuss in more detail hereinafter." born of the same parents' intracellular domain ", " tenuigenin structural domain " or the relational language that can exchange be meant and be in intracellular part in the polypeptide, it with those or be anchored on the cytolemma or the part that is exposed on the cell surface different.The heavy chain immunoglobulin polypeptide of film combining form comprises about three amino acid whose very short tenuigenin structural domains usually.Film combining form heavy chain immunoglobulin polypeptide of the present invention preferably comprises usually related with this heavy chain immunoglobulin film and the born of the same parents' intracellular domain of striding, for example with pre B cell in μ and the δ heavy chain be associated stride film and born of the same parents' intracellular domain, what perhaps be associated with any heavy chain immunoglobulin in the B memory cell strides film and born of the same parents' intracellular domain.But, can consider that also allos is striden film and born of the same parents' intracellular domain combines with given heavy chain immunoglobulin polypeptide, for example the μ heavy chain stride film and born of the same parents' intracellular domain can combine with born of the same parents' outside part of gamma heavy chain.Perhaps, that can use complete allogenic polypeptide strides film and/or cytoplasmic structure territory, for example main histocompatibility molecule, cell surface receptor, virus surface proteins matter stride film and cytoplasmic structure territory, embedded structure territory or composite structure territory.

Comprise in the embodiment of light chain polypeptide at those described immunoglobulin (Ig) subunit polypeptides, all included from any light chain immunoglobulin of any animal species.Described suitable in the literary composition and the preferred immunoglobulins light chain.Described light chain immunoglobulin comprises that from vertebrates such as bird (particularly chicken), fish and mammiferous light chain, wherein mammalian immune sphaeroprotein light chain is preferred.The example of light chain immunoglobulin comprises the people, mouse, dog, cat, horse, goat, rat, sheep, ox, pig, cavy and hamster light chain immunoglobulin.In these, the human normal immunoglobulin light chain is especially preferred.What it is also conceivable that is the heterozygosis light chain immunoglobulin, and it comprises the part from the light chain of one or more species, such as mouse/people's heterozygosis light chain immunoglobulin.The preferred immunoglobulins light chain comprises people κ and lambda light chain.Any a pair of light chain can be united same a pair of heavy chain and be formed immunoglobulin molecules, and it has distinctive H ₂L ₂Structure, this is well known to those of ordinary skill in the art.

Preferred aspect according to the present invention, each member in the polynucleotide library of describing in the literary composition (for example first polynucleotide library or the second polynucleotide library) comprises first nucleic acid molecule of the constant region for immunoglobulin that all members have in (a) encoded libraries, (b) second nucleic acid molecule of coding immune globulin variable region, wherein this second nucleic acid molecule is in the upstream of first nucleic acid molecule and meets its frame.Accordingly, preferably comprise the constant region for immunoglobulin that is associated with immune globulin variable region by the coded immunoglobulin (Ig) subunit polypeptide of each member in the polynucleotide of the present invention library light chain immunoglobulin or the heavy chain of such polynucleotide encoding (promptly by).

The constant region of light chain by " first nucleic acid molecule " coding comprises the only about half of of this subunit polypeptide, and is positioned at C-terminal, promptly is in the latter half of light chain polypeptide.Be called C in the literary composition _LThe constant region of light chain of constant region, C perhaps more specifically says so _κConstant region or C _λConstant region comprises by interchain disulfide bond and remains on about 110 amino acid in one " ring ".

By the constant region of heavy chain of " first nucleic acid molecule " coding comprise subunit polypeptide 3/4ths or more, and be positioned at C-terminal, promptly be in the latter half of heavy chain polypeptide.Be called C in the literary composition _HThe CH of constant region comprises about 110 amino acid whose 3 or 4 peptide rings or " structural domain ", and they are respectively sealed by interchain disulfide bond.More particularly, the CH of human normal immunoglobulin comprises C μ constant region, C δ constant region, C γ constant region, C α constant region and C ε constant region.C γ, C α and C δ heavy chain respectively contain 3 constant region structural domains, are commonly referred to C _H1, C _H2 and C _H3, and C μ and C ε heavy chain contain 4 constant region structural domains, are commonly referred to C _H1, C _H2, C _H3 and C _H4.The nucleic acid molecule of coding human normal immunoglobulin constant region can be easy to from deriving from the cDNA library of human B cell for example or their precursor, and by obtaining such as methods such as PCR, this is well-known to those skilled in the art, also has open in the following examples.

Immunoglobulin (Ig) subunit polypeptide of the present invention respectively contains an immune globulin variable region by " second nucleic acid molecule " coding.In the polynucleotide library, each polynucleotide comprises identical constant region, but that this library is contained is a plurality of, and promptly at least 2, preferably at least 10,100,10 ³, 10 ⁴, 10 ⁵, 10 ⁶, 10 ⁷, 10 ⁸Or 10 ⁹Individual different variable region.As well known to those of ordinary skill in the art, variable region of light chain is that each comprises light chain V by the nucleic acid molecule encoding of resetting _LThe district (is V specifically _κDistrict or V _λThe district) and light chain J district (be J specifically _κDistrict or J _λThe district).Similarly, variable region of heavy chain also is that each comprises heavy chain V by the nucleic acid molecule encoding of resetting _HDistrict, D district and J district.When being rearranged in cytodifferentiation, these occur in dna level.The nucleic acid molecule of encoding heavy chain and variable region of light chain can obtain from mature B cell and plasmocyte by for example PCR, and described cell finally breaks up, thereby express defined epitope is had specific antibody.In addition, if desired at the antibody of specific antigen, variable region this antigen immune of can using by oneself crosses, therefore produces that content enlarges can separate with plasmocyte with the mature B cell of the animal of the antibody variable region of this AI.Perhaps, more diversified library if desired can be from precursor cell, and for example pre B cell separates the variable region with immature B cells, because the rearrangement of immunoglobulin gene has taken place in these cells, but also is not exposed to self or non-autoantigen.For example, can from normal people's marrow of taking from a plurality of donors, separate the variable region by PCR.Perhaps, the variable region can be a synthetic, for example prepares by producing synthetic oligonucleotide in the laboratory, and perhaps deriving from kind is the manipulation in vitro of DNA, causes immunoglobulin gene rearrangement.

First and second nucleic acid molecule except encode respectively constant region for immunoglobulin and variable region, each member in polynucleotide of the present invention library further comprises the 3rd nucleic acid molecule of coded signal peptide as mentioned above, the 3rd nucleic acid molecule be located immediately at the coding variable region second nucleic acid molecule the upstream and conform to its frame.

" signal peptide " is meant the peptide sequence that for example can guide newborn immunoglobulin polypeptides subunit to be transported to host cell surface.Signal peptide also can be called " signal sequence " in the art, " leader sequence ", " secreting signal peptide " or " secretory signal sequence ".Signal peptide is typically expressed as a part complete or " prematurity " polypeptide, and generally is positioned at N-terminal.Common structure from the signal peptide of different proteins is described to positively charged n-district usually, but is subsequently-individual hydrophobic h-district and an electric neutrality polar c-district.In many cases, in case protein arrives its final destination, thereby the amino acid that comprises signal peptide is with regard to cut " maturation " form that produces this polypeptide.Cutting is by the enzyme institute catalysis that is called signal peptidase.(3 ,-1) rule declaration, for correctly carrying out of cutting, the residue of position-3 and-1 (relative cleavage site) must be little and be neutral.Referring to for example, McGeoch, Virus Res.3:271-286 (1985) and von Heinje, NucleicAcids Res.14:4683-4690 (1986).

All cells comprise host cell of the present invention, all have the Secretory Pathway of composing type, and the protein that will be output wherein comprises that excretory immunoglobulin (Ig) subunit polypeptide secretes from cell by this approach.These protein are processed approach by the ER-golgi body, and may modify at this.If detect less than other signals on the protein, it just is directed into cell surface and is secreted.Perhaps, the immunoglobulin (Ig) subunit polypeptide can finally become the inherent film component that is expressed in host cell surface.The immunoglobulin (Ig) subunit polypeptide of film combining form begins to follow the approach identical with secreted form, passes the ER chamber, but they are owing to the existence that stops encoding transport signals or " membrane spaning domain " rests in the ER film.Membrane spaning domain is the hydrophobic fragment of about 20 amino-acid residues, and they take alpha helical conformation when crossing over film.The protein that is embedded in the film is anchored in the phospholipid bilayer of cytoplasmic membrane.For secretory protein, there is a signal peptide in the N-terminal zone of transmembrane protein, and it can pass film and be cut when leaving the ER chamber.The form membrane of striding of heavy chain immunoglobulin polypeptide uses identical signal peptide with secreted form.

Signal peptide of the present invention can be the native immunoglobulin signal peptide, and is promptly coded by a part of sequence of natural heavy chain or light chain transcript; Or this sequence kept the guiding can handle the functional derivatives that the immunoglobulin (Ig) subunit polypeptide that is connected carries out the excretory ability with it.Perhaps, can use allos signal peptide or its functional derivatives.For example, can the choose signal peptide of tissue plasminogen activator or mouse beta-Glucuronidase replaces the natural signals peptide of immunoglobulin (Ig) subunit polypeptide.

The signal sequence of the membrane bound protein of known numerous species, membrane spaning domain and cytoplasmic structure territory.Can correspondingly use these sequences, promptly or will use (for example signal sequence and membrane spaning domain in couples from the described sequence of specified protein, perhaps signal sequence and cytoplasmic structure territory, perhaps membrane spaning domain and cytoplasmic structure territory), perhaps three are used together, perhaps use with each composition of taking from different proteins, perhaps, these sequences can be synthetic, derive from artificial delivery configuration territory consensus sequence as previously mentioned fully.

Especially preferred signal sequence and membrane spaning domain include, but are not limited to derive from CD8, ICAM-2, IL-8R, those of CD4 and LFA-1.Useful in addition sequence comprises some sequences like this, and they are from 1) the inherent membrane protein of I type, (residue 1-26 is a signal sequence, and 241-265 strides the film residue such as IL-2 acceptor β chain; Referring to Hatakeyama etc., Science244:551 (1989) and von Heijne etc., (residue 1-27 is a signal sequence, and 957-959 strides the film district, and 960-1382 is the cytoplasmic structure territory for Eur.J.Biochem.174:671 (1988) and insulin receptor β chain; Referring to Hatakeyama etc., the same and Ebina etc., Cell40:747 (1985))); 2) II type integral protein, (residue 29-51 strides the film district, and 2-28 is the cytoplasmic structure territory such as neutral endopeptidase; Referring to Malfroy etc., Biochem.Biophys.Res.Commun.144:59 (1987)); 3) type iii protein matter is such as human-cytochrome P450 NF25 (Hatakeyama etc., the same); And 4) IV type protein is such as people P-glycoprotein (Hatakeyama etc., the same).In this case, CD8 and ICAM-2 are especially preferred.For example the signal sequence of CD8 and ICAM-2 is positioned at the 5 ' end of transcription product.In the situation of CD8, signal sequence constitutes (Nakauchi etc., PNAS USA82:5126 (1985)) by amino acid/11-32, in the situation of ICAM-2, signal sequence constitutes (Staunton etc., Nature (London) 339:61 (1989)) by amino acid/11-21.These membrane spaning domains constitute (Nakauchi, the same) by amino acid/11 45-195 in CD8, constitute (Staunton, the same) by amino acid 224-256 in ICAM-2.

Perhaps, film anchoring structure territory comprises the GPI anchor, it forms covalent linkage by the glycosyl-phosphatidyl inositol key between described molecule and lipid bilayer, for example in DAF (referring to Homans etc., Nature333 (6170): 269-72 (1988), with Moran etc., J.Biol.Chem.266:1250 (1991)).In order to reach this situation, can be placed on 3 ' of immunoglobulin (Ig) or immunoglobulin fragment with striding the film sequence from the GPI sequence replacing of Thy-1.

Similarly, the myristylation sequence can be used as film anchoring structure territory.The myristylation of known c-src can make it raise on the plasma membrane.This is a kind of simple and effective film localization method, and condition is that proteinic preceding 14 amino-acid residues are responsible for this function fully (referring to Cross etc., Mol.Cell.Biol.4 (9) 1834 (1984); Spencer etc., Science262:1019-1024 (1993)).This primitive has demonstrated the location of reporter gene very effective, can be used for the zeta chain of grappling TCR.This primitive is placed on 5 ' of immunoglobulin (Ig) or immunoglobulin fragment, so that construct is navigated on the plasma membrane.Some other modification can be used for construct is anchored on plasma membrane such as palmitoylation; For example, the palmitoylation sequence of G protein coupling receptor kinases GRK6 sequence (Stoffel etc., J.Biol.Chem.269:27791 (1994)); The palmitoylation sequence of Visual purple (Barnstable etc., J.Mol.Neurosci.5 (3): 207 (1994)); And p21H-rasl albumen (Capon etc., Nature302; 33 (1983)).

Except first and second nucleic acid molecule of encode respectively constant region for immunoglobulin and variable region, each member in polynucleotide of the present invention as mentioned above library can comprise other nucleic acid molecule of the heterologous polypeptide of encoding in addition.These additional polynucleotide can be replenishing or surrogate the 3rd nucleic acid molecule of coded signal peptide.The additional nucleic acid molecule of this class coding heterologous polypeptide can be positioned at the upstream or the downstream of the nucleic acid molecule in coding variable chains zone (variable chain region) or heavy chain zone.

Heterologous polypeptide by additional nucleic acid molecule encoding can be a rescue sequence.So-called rescue sequence is can be used for immunoglobulin (Ig) or its fragment or polynucleotides encoding them are carried out purifying or isolating sequence.Therefore, for example peptide rescue sequence comprises the purifying sequence, such as the 6-His label that is used for Ni affinity post be used for the epi-position label of detection, immunoprecipitation or FACS (fluorescence-activated cell sorting).Suitable epi-position label comprises myc (using with commercially available 9E10 antibody), the BSP biotinylation target sequence of bacterial enzyme BirA, flu label, LacZ and GST.Described additional nucleic acid molecule can also the encoded peptide joint.

In a preferred embodiment, the heterologous polypeptide use that is combined.Therefore, for example can use the combination of any number of signal sequence, rescue sequence and critical sequences, and can be with or without joint sequence.People can be placed on 5 ' and 3 ' of immunoglobulin (Ig) or its segmental coded polynucleotide with the various fusion polynucleotides of coding heterologous polypeptide.It will be understood by those skilled in the art that these modules can use with many array modes and variation.

The polynucleotide that are included in first and second libraries are imported proper host cell.Proper host cell is characterised in that the immunoglobulin molecules that can express attached to their surface.Can described polynucleotide be imported host cell by method well-known to those skilled in the art.Suitable and preferred introduction method are disclosed in the literary composition.

Be understood that easily introduction method is according to the character that makes up the used carrier in polynucleotide library and difference.For example, can by lipofection for example (such as with anionic liposome (referring to for example Felgner etc., 1987 Proc.Natl.Acad Sci.USA84:7413), or cationic-liposome is (referring to for example Brigham, K.L. etc., Am.J Med Sci.298 (4): 278-2821 (1989); United States Patent (USP) 4897355 (Eppstein etc.)), electroporation, calcium phosphate precipitation (generally can be with reference to Sambrook etc., Molecular Cloning:ALaboratory Manual, second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989), protoplastis merges, and spheroplast merges, perhaps the DNA plasmid vector is imported host cell by the poly-a kind of farm tools method (Sussman etc., Cell.Biol.4:1641-1643 (1984)) in DEAE Portugal.Above document is all introduced herein as a reference in full.

When institute's choosing method is lipofection, nucleic acid and cationic-liposome can be formed mixture, cationic-liposome wherein comprises such as DOTMA:DOPE, DOTMA, DOPE, DC-cholesterol, DOTAP, Transfectam  (Promega), Tfx  (Promega), LipoTAXI ^TM(Stratagene), PerFect Lipid ^TM(Invitrogen), SuperFect ^TM(Qiagen).When nucleic acid is that this anionic liposome can wrap nucleic acid when carrying out transfection by anionic liposome.Preferably, adopt the experimental program of manufacturers (such as for Lipofectamine; Life Technologies Incorporated) imports DNA by liposome-mediated transfection.

At described plasmid is under the situation of virus vector, and to host cell importing most convenient is to infect by routine to carry out.But under many situations, can be by any method described above with the viral nucleic acid transfered cell, because viral nucleic acid is " infectivity ", after promptly viral nucleic acid imports in the cell, do not need other conditions, just enough make cell produce active progeny virion.But should be mentioned that, some viral nucleic acid, for example poxvirus nucleic acid is not infective, therefore must import with the supplementary component that is provided, for example by wrapping up the virion of viral nucleic acid, by being produced the cell of required viral element, perhaps pass through helper virus by through engineering approaches.

The described first and second polynucleotide libraries can or import host cell simultaneously by any order.For example, if the first and second polynucleotide libraries all make up in virus vector, no matter be infectivity or inactivation, carrier can import by infecting simultaneously with the form of mixture, and perhaps consecutive infection imports.If a library makes up in virus vector, another is in plasmid vector, most convenient be to import earlier a library, import another library again.

Behind first and second polynucleotide libraries importing host cell, make immunoglobulin molecules or its antigen-specific fragment on the film surface of described host cell, express or be secreted in the cell culture medium." making ... express " is to instigate the carrier that is imported into host cell to carry out transcribing and translating of the many skins of immunoglobulin (Ig) subunit, preferably makes host cell will assemble completely immunoglobulin molecules or its antigen-specific fragment is transported in film surface or the cell culture medium.Usually, make ... expression need cultivate the host cell that wherein imported polynucleotide under appropriate condition so that the carrying out of expressing.These conditions and expression required time change according to selected host cell and carrier, are well-known to those skilled in the art.

In certain embodiments, with those from the teeth outwards expressing immunoglobulin molecule the host cell that the solubility immunoglobulin molecules is secreted in the cell culture medium is contacted with antigen.In this article, " antigen " be can with any molecule of antibody, immunoglobulin molecules or its antigen-specific fragment specific combination." specific combination " is meant that antigen is attached on the CDR of antibody.Be " epi-position " with the interactional specifically part of CDR in the antigen, perhaps " antigenic determinant ".Antigen may comprise an epi-position, but as a rule, antigen comprises at least two epi-positions, and according to antigenic size, conformation and type, may comprise any amount of epi-position.

Antigen is peptide or polypeptide normally, but also can be any molecule or compound.For example, organic compound, for example dinitrophenol(DNP) or DNP, nucleic acid, no matter any mixture of carbohydrate or these compounds has or does not have peptide or polypeptide, can be suitable antigen.Minimum peptide or polypeptide epitope are considered to about 4 or 5 amino acid.Preferred peptide or polypeptide epitope comprise at least 7, and more preferably at least 9, most preferably at about 15 to 30 amino acid.Because CDR can discern the antigenic peptide or the polypeptide of tertiary structure, need not to be successive so constitute the amino acid of epi-position, in some situation, not even on same peptide chain.Among the present invention, peptide or polypeptide antigen preferably contain at least 4, and at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, the sequence that most preferably about 15 to 30 amino acid constitute.Peptide that preferably contains epi-position or be made up of epi-position or polypeptide are to the youthful and the elderly 10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95, or 100 amino-acid residues.Described antigen can be any form, also can be free, for example is dissolved in the solution, perhaps attached on any matrix.Suitable and preferred matrix is disclosed in the literary composition.In certain embodiments, as the following detailed description, antigen may be the part of the presenting cell of antigen expressed.

Should be appreciated that the method according to this invention can prepare is specific to any antigenic immunoglobulin molecules.Preferred antigen is " self " antigen, promptly derives from the antigen of same species with the immunoglobulin molecules that is produced.Give one example, may wish to prepare the people's antibody at human tumor antigen, described antigen includes, but are not limited to CEA antigen, GM2 antigen, Tn antigen, sTn antigen, Thompson-Friedenreich antigen (TF), Globo H antigen, Le (y) antigen, MUC1 antigen, MUC2 antigen, MUC3 antigen, MUC4, MUC5AC antigen, MUC5B antigen, MUC7 antigen, carcinomebryonic antigen, the β chain of human chorionic gonadotrophin (hCG β) antigen, HER2/neu antigen, PSMA antigen, EGFRvIII antigen, KSA antigen, PSA antigen, PSCA antigen, GP100 antigen, MAGE1 antigen, MAGE2 antigen, TRP1 antigen, TRP2 antigen, and tyrosine oxidase antigen.Other purpose " self " antigens include, but are not limited to cytokine, acceptor, part, glycoprotein and hormone.

The present invention comprises that also preparation is at the antigenic antibody on the infectant.The antigenic example of this class includes, but are not limited to bacterial antigens, virus antigen, parasite antigen and fungal antigen.The example of virus antigen comprises, but be not limited to adenovirus antigen, the α virus antigen, Calicivirus antigen, Calicivirus capsid antigen for example, coronavirus antigen, canine distemper virus antigen, Ebola virus antigen, enterovirus antigen, flavivirus antigen, hepatitis virus (A-E) antigen, for example hepatitis B core or surface antigen, simplexvirus antigen, for example hsv or varicella zoster virus glycoprotein antigen, immunodeficiency virus antigen, for example human immunodeficiency virus's coating or proteolytic enzyme antigen, infectious peritonitis virus antigen, influenza antigen, for example influenza A hemagglutinin or neuraminidase antigen, leukosis virus antigen, Marburg virus antigen, oncovirus antigen, orthomyxovirus antigen, papilloma virus antigen, parainfluenza virus antigen, hemagglutinin/neuraminidase antigen for example, paramyxovirus antigen, parvovirus antigen, pestivirus antigen, picornavirus antigen, for example bone marrow poliomyelitis virus capsid antigen, rabies virus antigen, rabies virus glycoprotein G antigen for example, reovirus antigen, retrovirus antigen, wheel virus antigen, and other carcinogenic or cancer is relevant virus antigens.

The example of bacterial antigens comprises, but be not limited to actinomycetes antigen, genus bacillus antigen, bacterioide antigen, Bordetella antigen, bartonia bodies antigen, burgdorferi antigen, B.bergdorferi OspA antigen for example, Brucella antigen, campylobacter antigen, carbonic acid gas is had a liking for Cellulomonas antigen, chlamydiaceae antigen, fusobacterium antigen, Corynebacterium antigen, Coxiella antigen, Dermatophilus antigen, enterococcus spp antigen, the ehrlichiosis body belongs to antigen, Escherichia antigen, Lang Xi Bordetella antigen not, Fusobacterium antigen, Haemobartonella antigen, hemophilus antigen, haemophilus influenzae type b outer membrane protein antigen for example, Helicobacterium antigen, Klebsiella antigen, L-type bacterial antigens, leptospira antigen, listeria spp belongs to antigen, Mycobacterium antigen, mycoplasma antigen, Neisseria antigen, Neorickettsia antigen, Nocardia antigen, pasteurella antigen, Peptococcus antigen, Peptostreptococcus antigen, Pn antigen, proteus antigen, Rhodopseudomonas antigen, Dermacentroxenus antigen, Luo Kali martensite belongs to antigen, salmonella antigen, Shigella antigen, Staphylococcus antigen, streptococcus antigen, streptococcus pyogenes M proteantigen for example, treponema antigen and Yersinia antigen, for example Yersinia pestis F1 and V antigen.

The example of fungal antigen includes, but are not limited to absidia antigen, Acremonium (Acremonium) antigen, Alternaria antigen, Aspergillus antigen, the mould genus antigen of frog excrement, Bipolaris antigen, budding yeast belongs to antigen, mycocandida antigen, ball spore Pseudomonas (Coccidioides) antigen, the mould antigen of ear, Cryptococcus antigen, Curvularia antigen, Epidermophyton antigen, the mould genus antigen of outer blank handle, Geotrichum antigen, Histoplasma antigen, Madurella antigen, Malassezia antigen, Microsporon antigen, Moniliella antigen, genus mortierella antigen, Mucor antigen, paecilomyces antigen, Penicillium antigen, Phialemonium antigen, Saksenaea antigen, Prototheca antigen, Pseudallescheria antigen, Pseudomicrodochium antigen, pythium antigen, rhinosporidium seeberi belongs to antigen, Rhizopus antigen, Scolecobasidium antigen, Sporothrix antigen, Stemphylium antigen, Trichophyton antigen, Trichosporon antigen and Xylohypha antigen.

The antigenic example of protozoon parasite comprises, but be not limited to Babesia antigen, Balantidium antigen, bass Eimeria (Besnoitia) antigen, Cryptosporidium (Cryptosporidium) antigen, Eimeria (Eimeri) antigen a antigen, brain occupies Eimeria (Encephalitozoon) antigen, Endamoeba antigen, Giardia antigen, Hammondia antigen, Hepatozoon (Hepatozoon) antigen, Deng spore Eimeria antigen, Leishmania antigen, microsporidium belongs to (Microsporidia) antigen, and neospora belongs to (Neospora) antigen, Nosema antigen, Pentatrichomonas antigen, plasmodium antigen, P.falciparum circumsporozoite (PfCSP) for example, sporozoite surface protein 2 (PfSSP2), liver attitude (liver state) antigen 1 carboxyl terminal (PfLSA-1 c-term), with outward transport albumen 1 (PfExp-1) antigen, pneumocystis (Pneumocystis) antigen, Miescheria antigen, Schistosoma (Schistosoma) antigen, Theileria (Theileria) antigen, toxoplasma antigen and trypanosoma antigen.

The antigenic example of helminthism worm comprises, but be not limited to Acanthocheilonema (Acanthocheilonema) antigen, Aelurostrongylus antigen, Ancylostoma antigen, Angiostrongylus antigen, Ascaris antigen, cloth Shandong Turbatrix (Brugia) antigen, Bunostomum (Bunostomum) antigen, Hepaticola antigen, Chabertia belongs to (Chabertia) antigen, Cooperia (Cooperia) antigen, Crenosoma (Crenosoma) antigen, four buttock line Eimeria (Dictyocaulus) antigens, Dioctophyma (Dioctophyme) antigen, bivalve Turbatrix (Dipetalonema) antigen, two Phyllobothriums (Diphyllobothrium) antigen, Diplydium antigen, Dirofilaria antigen, Dracunculus (Dracunculus) antigen, Enterobius antigen, Filaroides antigen, Haemonchus (Haemonchus) antigen, harelip Ascaris (Lagochilascaris) antigen, sieve Ah Turbatrix (Loa) antigen, Mansonella antigen, Muellerius belongs to (Muellerius) antigen, dwarf's body belongs to (Nanophyetus) antigen, Necator antigen, Nematodirus (Nematodirus) antigen, oesophagostomum antigen, Onchocerca antigen, Opisthorchis antigen, this off-line Eimeria (Ostertagia) antigen difficult to understand, by-pass Eimeria (Parafilaria) antigen, and grow genus antigen, parascris antigen, physaloptera antigen, Protostrongylus antigen, Setaria (Setaria) antigen, Spirocerca (Spirocerca) antigen, winding palace belongs to (Spirometra) antigen, Stephanofilaria antigen, Strongyloides antigen, Strongylus antigen, Thelazia (Thelazia) antigen, Belascaris (Toxascaris) antigen, bend first Turbatrix antigen, Trichinella antigen, trichostrongylus antigen, Trichocephalus antigen, hook Turbatrix antigen and Wuchereria antigen.

In those immunoglobulin molecules are expressed in selection and screening scheme on the host cell surface, host cell of the present invention is contacted with antigen by such method, the antigen of CDR that this method makes specific recognition be expressed in the immunoglobulin molecules of host cell surface combines with CDR, comes thereby make with antigen-specific bonded host cell and the antigenic host cell difference of those debonds.The method that any host cell that can make the antigen expressed specific antibody contacts with antigen all comprises in the present invention, for example, if host cell suspends, antigen is attached on the solid substrate, then will be captured on the solid substrate with antigen-specific bonded cell, thereby make that antigenic those cells of debond can be washed off, can reclaim subsequently in conjunction with cell.Perhaps, if host cell attached on the solid substrate, by specific combination antigen, causes these cells to discharge (for example, because necrocytosis) from matrix, then can from cell conditioned medium liquid, reclaim them.Disclose the preferred method that host cell of the present invention is contacted with antigen in the literary composition, especially utilized by three molecular recombination and be structured in library in the vaccinia virus vector.

At a screening method that preferably is used for detecting the antigen specific immune globulin molecule that is expressed on the host cell surface, with host cell of the present invention with selecting the antigen incubation, the direct mark of wherein said antigen fluorescein-5-isothiocyanic acid (FITC), perhaps the indirect labelling vitamin H detects with the strepto-affinity of FITC mark is plain then.Other fluorescent probes that can adopt those skilled in the art to be familiar with.In the incubation process, the selection antigen that mark is good combines with the antigen specific immune globulin molecule.Can select the cell of expression by fluorescence activated cell sorting, thereby make and to distinguish with antigen-specific bonded host cell and the antigenic host cell of debond at the antibody receptor of specific fluorescent labeled antigen.Along with can sorting 1 * 10 in 1 hour ⁸The appearance of the cell sorter of individual above cell just can be screened a large amount of cells in the recombinant vaccinia library of having infected immunoglobulin gene, thereby selects expression at the cell subsets of selecting antigenic specific antibody acceptor.

Behind results and the antigen-specific bonded host cell, from these host cells, reclaim the polynucleotide in first library." recovery " is meant roughly required composition and unwanted component separating opened.For example, the disengaging from the solid substrate comes " recoverys " and antigen bonded host cell according to host cell, by with the roughing out of other cellular constituents from these cells the polynucleotide in " recovery " first library.Should be mentioned that term " recovery " do not represent the purifying of any kind or separate to remove virus removal and other compositions.The recovery of polynucleotide can be undertaken by the known any ordinary method of those of ordinary skills.A preferred aspect, described polynucleotide by results infectious viral particle reclaim, for example, wherein made up the vaccinia virus vector particle in first library, they be included in antigen bonded host cell in.

At some immunoglobulin molecules by fully from the screening scheme that host cell surface is secreted out, wherein cultivating the cell culture medium in host cell pond, i.e. " conditioned medium ", can carry out " contact " with antigen by a kind of like this method, this method makes the antigen of CDR of specific recognition immunoglobulin molecules combine with CDR, and can detect antigen-antibody interaction.These class methods include, but are not limited to immunoblotting, ELISA assay method, RIA assay method, RAST assay method and immunofluorescence assay.Perhaps, described conditioned medium is carried out the functional examination of specific antibodies.The example of this class assay method comprises, but be not limited to viral neutralisation (being used for antibody) at specific virus, detection method (being used for the antibody at specific bacteria) is nursed one's health/engulfed to bacterium, antibody-dependent cytotoxicity (ADCC) detection method, detection is to the inhibition or the promoted assay method of some cell function, method, hemagglutination test and the hemagglutination-inhibition test of the histamine release of the IgE mediation of detection mastocyte.These measuring methods can detect the antigen-specific antibodies with required function feature.

After identifying the conditioned medium of the immunoglobulin molecules that contains conjugated antigen specifically or have required functional character, further screen step, up to being recovered to the host cell that produces the target immunoglobulin molecules, from these host cells, reclaim the polynucleotide in first library then.

Those of ordinary skills are readily appreciated that, the polynucleotide of identification code immunoglobulin (Ig) subunit polypeptide may need two or the above-mentioned selection of more wheels, must carry out two or the above-mentioned screening of more wheels.One takes turns the polynucleotide that selection not necessarily can be separated to the required first immunoglobulin (Ig) subunit polypeptide of pure coding; The mixture that obtains after the first round selection may be rich in required polynucleotide, but also pollute the impurity insertion sequence is arranged.The sieve method of describing in the literary composition can identify the pond of containing active host cell and/or immunoglobulin molecules, but nonactive kind is also contained in these ponds.Therefore, active pond is further segmented, and carries out other several screenings of taking turns.So the polynucleotide of the identification code first immunoglobulin (Ig) subunit polypeptide (it and the second immunoglobulin (Ig) subunit polypeptide can form target immunoglobulin molecules or its antigen-specific fragment when uniting) may need several selections of taking turns and/or screening or more favourable, have so just improved the ratio of the cell that contains required polynucleotide.Correspondingly, the polynucleotide that the further requirement of this embodiment will be recovered to the first round import second group of cell, and carry out second and take turns selection.

Therefore, aforesaid first select step may maybe must be repeated 1 or more times so that the polynucleotide of enrichment coding purpose immunoglobulin (Ig) subunit polypeptide.In order to repeat first step of this embodiment, polynucleotide or the polynucleotide pond that is recovered to as mentioned above imported the host cell group, these cell group energys are expressed the immunoglobulin molecules by the polynucleotide encoding in the library.These host cells can be identical with the cell type of use in the first round selection, and perhaps different, condition is to express described immunoglobulin molecules.The second polynucleotide library is also imported these host cells, immunoglobulin molecules or its antigen-specific fragment are expressed in the surface of cell membrane of described host cell or cell culture medium.Similarly, cell or conditioned medium are contacted with antigen, perhaps in the functional assays method, detect this substratum, once more from expressing those cells or the concentrated polynucleotide that reclaim first library of host cell that combine and/or have the immunoglobulin molecules of required function characteristic with antigen-specific.These steps can be repeated one or repeatedly, make polynucleotide be able to enrichment from first library, described polynucleotide encoding immunoglobulin (Ig) subunit polypeptide, it is energy specific combination antigen and/or has the part of the immunoglobulin molecules of required function characteristic, or its antigen-specific fragment.

To carry out from the polynucleotide of interest in first library after the suitable enrichment as mentioned above, these polynucleotide that are recovered promptly are " isolating ", be that they break away from from its natural surroundings basically, in most of and the library not the polynucleotide of coding for antigens specific immunoglobulin subunit polypeptide separated.For example, with regard to purpose of the present invention, the clone's polynucleotide that are included in the carrier are considered to isolating.Should be understood that,, can be recovered to two or more different immunoglobulin (Ig) subunit polypeptides of energy specific combination same antigen by the method for describing in the literary composition.Accordingly, coding also is considered to " isolating " with the polynucleotide mixture of same antigen bonded polypeptide.Other examples of isolating polynucleotide comprise those remain in the heterologous host cell or solution in the dna molecular of (part or basically) purifying.But with regard to purpose of the present invention, the polynucleotide that are included among such clone are not " isolating ", these clones are the members that mix the library, and for example do not open with other clone and separate in the library owing to the many skins of coding for antigens specific immunoglobulin subunit.For example, the polynucleotide that are included in the virus vector are " isolating " reclaiming and carrying out behind the plaque purifying, and the polynucleotide that are included in the plasmid vector are " isolating " after amplification from single bacterial colony.

Known a kind of antigen can comprise two or more epi-positions, any one epi-position may be in conjunction with several different immunoglobulin molecules, therefore expection can be recovered to several suitable polynucleotide by the first step of this embodiment, for example 2,3,4,5,10,100 or the polynucleotide of more a plurality of immunoglobulin (Ig) subunit polypeptides of all encoding, described subunit polypeptide can form and purpose antigen-specific bonded immunoglobulin molecules or its antigen-specific fragment when combining with the coded suitable immunoglobulin (Ig) subunit polypeptide of the polynucleotide in second library.Each different polynucleotide that expection is reclaimed from first library can be separated individually.But these polynucleotide may have a group polynucleotide of the specific polypeptide of same antigen and are separated as coding, and these polynucleotide may be " isolating " together.No matter this class polynucleotide mixture is isolating or isolating jointly separately, can be as explained later, separately or with 2,3,4,5,10,100 or more a plurality of polynucleotide pond import host cell together in second goes on foot.

In case from first library, be separated to one or more suitable polynucleotide, in second step of this embodiment, identify one or more polynucleotide, but the immunoglobulin (Ig) subunit polypeptide of their codings can be united formation specific combination purpose antigen or have immunoglobulin molecules or its antigen-specific fragment of required function characteristic with separating from the coded immunoglobulin (Ig) subunit polypeptide of the polynucleotide in first library.

Accordingly, second step comprised that the second polynucleotide library with the coding second immunoglobulin (Ig) subunit polypeptide imported the host cell group of energy expressing immunoglobulin molecule, import at least one to identical host cell group and separate polynucleotide as mentioned above from first library, make in described host cell surface expressing immunoglobulin molecule or its antigen-specific fragment, perhaps be secreted in the cell culture medium fully, the conditioned medium of these host cells or host cell growth is contacted with the purpose specific antigen, perhaps conditioned medium is carried out Function detection, but then from the antigenic host cell of those binding purposess or during showing required active substratum, reclaim the polynucleotide in second library in the host cell grown.Therefore second step and the first step are very similar, and just the second coded immunoglobulin (Ig) subunit polypeptide of polynucleotide in second library separates the polynucleotide combination in host cell from first library with those.As above-mentioned, can use the single clone polynucleotide of separation from first library, perhaps can import the pond of separation simultaneously from several polynucleotide in first library.Identical with the above-described the first step, carry out one or severally take turns enrichment, promptly diminishing pond is selected or screened, thereby make in second library polynucleotide of the coding second immunoglobulin (Ig) subunit polypeptide be able to enrichment, the described second immunoglobulin (Ig) subunit polypeptide is can specific combination purpose antigen or show the part of the immunoglobulin molecules of required function characteristic.Same identical with the first step, from second library, separate one or more required polynucleotide.If used separation polynucleotide pond in the enrichment second step several wheel the early, preferably in enriching step subsequently, utilize and separate from the littler polynucleotide pond in first library, perhaps more preferably utilize the single clone's polynucleotide that separate from first library.For any separation from first library and be used for the single polynucleotide of the chosen process of the second library polynucleotide, might from second library, be separated to several (promptly 2,3,4,5,10,100 or more a plurality of) polynucleotide of the coding second immunoglobulin (Ig) subunit polypeptide, but the described second immunoglobulin (Ig) subunit polypeptide can be united formation specific combination purpose antigen or show the immunoglobulin molecules of required function characteristic with separating from the first coded immunoglobulin (Ig) subunit polypeptide of the polynucleotide in first library, or its Fab.

The selection/screening method in library for the coding single-chain fragment only needs a library, rather than first and second libraries, and only needs 1 selection/screening step.Similar with any step of the two-step approach of immunoglobulin (Ig), a this step carries out two-wheeled in the selection/sieve method or the more wheels enrichment also may be more favourable.

Carrier.When in eukaryotic cell, making up antibody library, can use any conventional carrier that can in eukaryotic cell, express.For example, can in virus, plasmid, phage or phagemid carrier, make up the library, as long as selected concrete carrier comprises transcribing and the translational control district of playing a role in eukaryotic cell.But, preferably in the eucaryon virus vector, make up aforesaid antibody library.

The eucaryon virus vector can be an any kind, for example animal virus vector or plant viral vector.The natural gene group of virus vector can be normal chain, minus strand or double-stranded RNA, and perhaps DNA, and natural gene group can be an annular or linear.For animal virus vector, comprise that those infect the virus vector of invertebratess (for example insect, protozoon or helminthism worm) or vertebrates (for example Mammals, bird, fish, Reptilia and Amphibians).Select virus vector only to be subjected to maximum restriction of inserting fragment and protein expression level.Suitable virus vector is that those infect yeast and other fungal cells, insect cell, protozoan cell, vegetable cell, bird cell, fry cell, the Reptilia cell, the virus vector of Amphibians cell or mammalian cell, special preferred mammal virus vector.Any conventional virus vector may be used to the present invention, include but not limited to poxvirus vector (for example vaccinia virus), herpesvirus vector (for example hsv), adenovirus carrier, baculovirus vector, retroviral vector, picornavirus carrier (for example poliovirus), Alphavirus carrier (for example sindbis virus) and enterovirus (for example encephalomyocardis virus (mengovirus)).Preferred dna viral vector, for example poxvirus, simplexvirus, baculovirus and adenovirus.Just as described in detail below, preferred especially poxvirus, especially vaccinia subgroup virus, particularly vaccinia virus.In a preferred embodiment, use the host cell that can produce the infectious viral particle of any selected virus vector.Many conventional virus vector such as vaccinia virus, have the host range of non-constant width, therefore can use many different host cells.

As above-mentioned, can in identical or different carrier, make up first and second libraries of the present invention.But, in preferred embodiments, first and second libraries are prepared to the polynucleotide that can reclaim first library and second library easily, for example in the first step, the polynucleotide in first library are separated with the polynucleotide in second library easily, in second step, from the polynucleotide in first library, reclaimed the polynucleotide in second library easily.For example, in the first step, if make up first library in virus vector, second library construction is in plasmid vector, can be recovered to the polynucleotide in first library at an easy rate with the form of infectious viral particle, and the polynucleotide and the cell debris in remaining second library.Similarly, in second step, if second library construction in virus vector, and the polynucleotide in first library that is separated in the first step are imported into plasmid vector, then can easily be recovered to the infectious viral particle that contains the second library polynucleotide.

Polynucleotide from first library are imported in the host cell in plasmid vector when the second polynucleotide library or separation, the coded immunoglobulin (Ig) subunit polypeptide of polynucleotide that comprises in then preferred this plasmid vector has operably connected transcription regulatory region, and described control region is driven by the coded protein of the virus vector that contains other libraries.For example, if in poxvirus vector, make up first library, in plasmid vector, make up second library, the polynucleotide that preferably are structured in the coding second immunoglobulin (Ig) subunit polypeptide in the plasmid library operably connect the transcription regulatory region that can play a role, preferred promoter in the tenuigenin of the cell of poxvirus infection.Similarly, in second step, the polynucleotide that will separate if desired from first library insert plasmid vector, in poxvirus vector, make up second library, then preferable separation is from first library and be inserted into polynucleotide in the plasmid and operably connect the transcription regulatory region that plays a role in can the tenuigenin at the cell of poxvirus infection, preferred promoter.Suitable and the preferred example of this class transcription regulatory region is disclosed in the literary composition.Like this, the polynucleotide in second library have only also infected the cell inner expression of poxvirus at those.

Yet, if can in a virus vector, keep first and second libraries, and separate those polynucleotide from first library, and will 1 or these two libraries maintain in two kinds of different carrier systems, will be very easily.Therefore, among the present invention, can be to maintaining first in the virus vector or the second library sample deactivation, thereby make the viral vector infection cell, the genome of virus vector is transcribed, but carrier does not duplicate, promptly when described virus vector transfered cell, the gene product that this viral genome is carried, for example the immunoglobulin (Ig) subunit polypeptide obtains expressing, but does not produce infectious viral particle.

One preferred aspect, by handle the library sample that is structured in the virus vector with 4 '-aminomethyl-Trisoralen (psoralene), then virus vector is exposed to ultraviolet ray (UV), makes the first or second library inactivation that is structured in the eucaryon virus vector.Psoralene and UV inactivation of viruses are well known to those of ordinary skill in the art.Referring to, Tsung for example, K. etc., J.Virol.70:165-171 (1996), this article is incorporated herein by reference in full.

Psoralene is handled and to be generally included the psoralene incubation that the acellular sample of virus vector and concentration is arrived about 20 μ g/ml at about 0.1 μ g/ml, the concentration of preferred psoralene is that about 1 μ g/ml arrives about 12.5 μ g/ml to the about 2.5 μ g/ml of about 17.5 μ g/ml ' to the about 5 μ g/ml of about 15 μ g/ml ', about 7.5 μ g/ml are to about 12.5 μ g/ml, and perhaps about 9 μ g/ml are to about 11 μ g/ml.Therefore, the concentration of psoralene can be about 0.1 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, 2 μ g/ml, 3 μ g/ml, 4 μ g/ml, 5 μ g/ml, 6 μ g/ml, 7 μ g/ml, 8 μ g/ml, 9 μ g/ml, 10 μ g/ml, 11 μ g/ml, 12 μ g/ml, 13 μ g/ml, 14 μ g/ml, 15 μ g/ml, 16 μ g/ml, 17 μ g/ml, 18 μ g/ml, 19 μ g/ml, or 20 μ g/ml.Preferably, the concentration of psoralene is about 10 μ g/ml.With herein, the time considered in term " approximately ", chemical substance concentration, temperature, pH and other usually in the laboratory or the factor of factory be difficult to accurately, may in some amount, change according to the type of measuring and the equipment that is used to measure.

Usually will carry out for some time with the incubation of psoralene at the UV pre-irradiation.Time period is preferably UV pre-irradiation about 1 minute to about 20 minutes.Preferably, time period changed in about 2 minutes to about 19 minutes, from about 3 minutes to about 18 minutes, from about 4 minutes to about 17 minutes, from about 5 minutes to about 16 minutes, from about 6 minutes to about 15 minutes, from about 7 minutes to about 14 minutes, from about 8 minutes to about 13 minutes, perhaps from about 9 minutes to about 12 minutes.Therefore, the incubation time can be about 1 minute, about 2 minutes, and about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, perhaps about 20 minutes.More preferably, carry out 10 minutes incubations at the UV pre-irradiation.

To be exposed to UV light through the virus that psoralene was handled then.UV can be any wavelength, but preferred long wave UV light, for example about 365nm.Be exposed to UV and will carry out about 0.1 minute to about 20 minutes time range.Preferably, time range was at about 0.2 to about 19 minutes, and about 0.3 arrives about 18 minutes, about 0.4 to about 17 minutes, about 0.5 to about 16 minutes, about 0.6 to about 15 minutes, about 0.7 to about 14 minutes, about 0.8 to about 13 minutes, about 0.9 to about 12 minutes, about 1 to about 11 minutes, about 2 to about 10 minutes, about 2.5 to about 9 minutes, about 3 to about 8 minutes, about 4 to about 7 minutes, perhaps about 4.5 to about 6 minutes.Therefore, the incubation time can be about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, perhaps about 20 minutes.More preferably, virus vector was exposed to UV wide about 5 minutes.

In eukaryotic cell, assemble and expressing immunoglobulin molecule or the segmental ability of its antigen-specific by two polynucleotide libraries of coding immunoglobulin (Ig) subunit polypeptide, than the method for manufacture order chain antibody in bacterial system significant raising has been arranged, selected to have each species specific immunoglobulin molecules or the segmental basis of its antigen-specific because two step chosen processs can be used as.

Following embodiment provides the example of specific embodiments, and they are further illustrated but do not limit the present embodiment.As what describe in detail previously, divide two stages to finish to the specific immunoglobulins subunit polypeptide for example selection of heavy chain immunoglobulin and light chain.At first, in eucaryon virus vector (for example poxvirus vector), make up from natural or through the celliferous diversified heavy chain of the immunoglobulin (Ig) of immune donor library; Similar diversified light chain immunoglobulin library construction is in plasmid vector, and wherein the expression of recombination is subjected to the regulation and control of viral promotors, perhaps is structured in to handle in the eucaryon virus vector of deactivation by for example psoralene and UV.Viral vector infection with the encoding heavy chain library of about 1 (MOI=1) of infection multiplicity can expressing immunoglobulin molecule or the segmental host cell of its antigen-specific." infection multiplicity " refers to infect the mean number of the virion of each host cell.For example, MOI is 1 infection if desired, and promptly each cell is on average infected by 1 virion, and just the number of the infectious viral particle that will be used to infect is adjusted to the number that equals to treat cells infected.

According to this strategy,,, perhaps use the light chain virus library host cells infected of deactivation with light chain plasmid library transfection host cell making each cell can absorb and express under the condition of polynucleotide of average 10 or more a plurality of coding light chain polypeptides.Under such condition, an independent host cell can be expressed panimmunity globulin molecule or its fragment, the H of different light chains and identical heavy chain constitutive characteristic in each host cell ₂L ₂Structure.

Those of ordinary skill in the art will appreciate that, it is difficult controlling the plasmid number that cell absorbs, and induce competence because successful transfection depends in cell, and this is not unified, may cause absorbing the DNA of different amounts.Therefore, carefully control in those embodiments of polynucleotide number in second library that imports in each host cells infected, preferably use the inactivation of viruses carrier at needs because this moment virus the easier control of infection multiplicity.

At the multiple light chain of single host cell inner expression, unite a heavy chain, the effect of the affinity (" avidity ") that reduces the specific antigen immunoglobulin (Ig) is arranged, but useful for selecting than the binding site of high affinity (" affinity ").With in the text, term " avidity " is meant the criterion of the intensity of single epi-position binding domain-immunoglobulin molecule CDR.Referring to for example Harlow 27-28 page or leaf.With in the text, term " affinity " total refer to the immunoglobulin (Ig) group and mixture that antigen forms stability, be exactly immunoglobulin mixture and antigenic functional bonding strength.Referring to for example Harlow 29-34 page or leaf.Single immunoglobulin molecules in " affinity " and the colony is relevant with the avidity of defined epitope, also with the immune globulin bletilla this antigenic tire relevant.For example, divalence monoclonal antibody and an interaction that has between the antigen (such as polymer) that highly repeats the epi-position structure just possess high-affinity.Those of ordinary skills can both understand, if host cell is at its surface expression immunoglobulin molecules, each all comprises specific heavy chain, but lip-deep different immunoglobulin molecules comprises different light chains, and then this host cell will descend to given antigenic " affinity ".Yet the possibility that reclaims such a group immunoglobulin molecules has but improved, and the dependency of these immunoglobulin molecules is to comprise the common heavy chain, but by uniting with different light chains, their energy and specific antigen are with the avidity reaction of certain limit.Correspondingly, by adjust different light chains or its fragment number of (they can be united with heavy chain or its fragment of some amount) in given host cell, the invention provides immunoglobulin molecules or the segmental method of its antigen-specific that a kind of selection and enrichment have various avidity levels.

When in the first step of aforesaid selection immunoglobulin molecules or the segmental method of its antigen-specific, utilizing this strategy, preferably in the eucaryon virus vector, make up first library, with the first library host cells infected of MOI, allowing each infected host cell picked-up nearly to import second library under the condition of the polynucleotide in 20 second libraries at about 1 to 10 (preferably approximately 1).For example, if second library construction is in the inactivation of viruses carrier, with MOI at about 1 to 20 the second library host cells infected, though different according to the virus vector that uses with the characteristic of purpose immunoglobulin molecules, may need to be higher or lower than the MOI of this scope.If in plasmid vector, then regulating transfection conditions, second library construction make 0 to 20 plasmid enter each host cell.By increasing or reduce the average number of the second library polynucleotide that allow to enter each infected cell, control and select the original higher or lower affinity reaction of antagonism.

More preferably, when first library construction in virus vector, with MOI at about 1-9, about 1-8, about 1-7, about 1-6, about 1-5, about 1-4, or the about first library host cells infected of 1-2.In other words, host cell is infected by first library with MOI about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2 or about 1.Most preferably, with MOI be about 1 the first library host cells infected.

When second library construction is in plasmid vector, preferably can absorb and reach about 19 at each infected host cell of permission, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, under the condition of about 2 or about 1 second library polynucleotide, plasmid vector is imported host cell.Most preferably, when second library construction is in plasmid vector,, plasmid vector is imported host cell allowing each infected host cell to absorb under the condition that reaches about 10 second library polynucleotide.

Similarly, when in the virus vector of second library construction in deactivation, more preferably with the about 1-19 of MOI, about 2-18, about 3-17, about 4-16, about 5-15, about 6-14, about 7-13, approximately 8-12 or approximately 9-11 second library is imported host cell.In other words, host cell is about 20, about 19, about 18, about 17, about 16, about 15 by MOI, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or infect in about 1 second library.One most preferred aspect, be about 10 usefulness, second library host cells infected with MOI.Those of ordinary skills can understand, and the titre of inactivation of viruses and " MOI " can't directly measure, but can infer from the titre of infectious virus liquid storage (they are inactivated subsequently) of beginning and this titre.

One of the present invention most preferred aspect, first library construction is in virus vector, second library construction is about 1 the first library host cells infected with MOI in the virus vector of deactivation, MOI is about 10 the second library host cells infected.

Among the present invention, preferred virus vector is derived from poxvirus, for example vaccinia virus.First library construction of the first immunoglobulin (Ig) subunit polypeptide is in poxvirus vector if encode, be subjected to the regulation and control of poxvirus promotor by the expression of the second immunoglobulin (Ig) subunit polypeptide that is structured in second library coding in plasmid vector or the inactivation of viruses carrier, then the second immunoglobulin (Ig) subunit polypeptide obtains high level expression in the tenuigenin of the cell of poxvirus infection, need not nuclear and integrates.

In second step of aforesaid selection immunoglobulin (Ig), preferably with second library construction in infectious eucaryon virus vector, be about 1 to 10 the second library host cells infected with MOI.More preferably, when second library construction in virus vector, be about 1-9 with MOI, about 1-8, about 1-7, about 1-6, about 1-5, approximately 1-4 or the approximately second library host cells infected of 1-2.In other words, host cell is about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2 or about 1 second library infection by MOI.Most preferably, with MOI be about 1 the second library host cells infected.

In second step of selecting immunoglobulin (Ig), separated from the polynucleotide in first library.In certain embodiments, with the single first library polynucleotide, promptly the clone imports the host cell that is used to separate the second library polynucleotide.In this case, allowing each host cell to have under the condition of about at least 1 polynucleotide to import host cell with separating polynucleotide from first library.But because all polynucleotide from first library of being imported into all are identical, promptly by the copy of clone's polynucleotide, so it is not too important to import the quantity of the polynucleotide in any given host cell.For example, if clone's the polynucleotide of separation from first library are included in the inactivation of viruses carrier, this carrier will be about 1 to be imported into MOI, but MOI also is feasible greater than 1.Similarly, if clone's the polynucleotide of separation from first library are imported into plasmid vector, the number of plasmid that then imports any given host cell is inessential, but should regulate transfection conditions to guarantee having imported at least 1 polynucleotide in each host cell.If for example from first library, be separated to several different polynucleotide, can adopt or embodiment.In this embodiment, separation advantageously can be imported the host cell that has infected the second polynucleotide library from the pond of two or more different polynucleotide in first library.In this situation, if the polynucleotide that separate from first library are included in the inactivation of viruses carrier, the MOI of preferred inactivated virus particle is greater than about 1, for example about 2, about 3, about 4, about 5 or higher; If perhaps the polynucleotide that separate from first library are included in the plasmid vector, the condition that preferred permission about at least 2,3,4,5 or more a plurality of polynucleotide enter each cell.

Poxvirus vector.As above-mentioned, being preferred for virus vector of the present invention is poxvirus vector." poxvirus " comprises all members of Poxviridae, comprises subfamily Chordopoxviridae (vertebrates poxvirus) and Entomopoxviridae (entomopoxvirus).Referring to, B.Moss (Virology, 2dEdition, B.N.Fields, D.M.Knipe etc., Eds., Raven Press, 2080 (1990)) for example.Vertebrates poxvirus (Chordopoxviruses) comprises with subordinate etc.: vaccinia subgroup virus (for example vaccinia virus, variola virus, raccoonpox virus); Fowlpox virus (for example fowlpox virus); Goat capripoxvirus (for example sheep pox virus), hare poxvirus (for example rabbit (Shope) fibroma and myxoma); And pig pox virus (for example pig pox virus).Entomopoxvirus comprises three genus: A, B and C.Among the present invention, preferred vaccinia subgroup virus.Vaccinia virus is the prototype vaccinia subgroup virus, has been set up as the proteinic carrier of expressing heterologous, and it has been had characteristic research well.Among the present invention, preferred vaccinia virus vector, especially those are established the vaccinia virus vector that carries out three molecular recombination.But other vaccinia subgroup viruses, especially raccoonpox virus also are developed to carrier, and better characteristic is arranged in some applications.

The characteristics of poxvirus are that volume is big, and are very complicated, and contain similarly big and complicated genome.What deserves to be mentioned is that poxviral replication carries out fully in the kytoplasm of host cell.The middle body of poxvirus genome group is all similar, and end is more changeable.Therefore, the middle body of poxvirus genome group is considered to carrying the gene of the total critical function of responsible all poxvirus, such as duplicating.On the contrary, the terminal portions of poxvirus genome group as if be responsible for that those change between different poxvirus such as features such as pathogenic and host ranges, and more may be virus in tissue culture, duplicate unessential.Therefore infer, if desire is passed through the rearrangement or the removal of dna fragmentation or is imported exogenous dna fragment and the modified vaccinia genome, then be usually located at the part that those foreign DNAs that are rearranged, remove or be imported into of far-end interrupt in the n DNA and preferably be in more distal area, because estimate that they are to virus replication and the generation of infectious virion and nonessential in tissue culture.

Natural vaccinia virus gene group is crosslinked double-stranded linear DNA molecule, and about 186000 base pairs are arranged, and is characterised in that inverted terminal repeat sequence.The genome of vaccinia virus is all checked order, but the function of most gene products or the unknown.Goebel, S.J. etc., Virology179:247-266,517-563 (1990); Johnson, G.P. etc., Virology196:381-401.In the vaccinia virus gene group, identify a large amount of nonessential regions.Referring to for example, Perkus, M.E. etc., Virology152:285-97 (1986); And Kotwal, G.J. and Moss B., Virology167:524-37.

Be used to express in those embodiments of immunoglobulin (Ig) subunit polypeptide at poxvirus vector, especially vaccinia virus vector, can use any suitable poxvirus vector.Preferred immunoglobulin subunit polypeptide library be carried in the carrier for the growth of carrier and duplicate and nonessential zone in, therefore can produce infectious virus.Though the nonessential region of many vaccinia virus gene groups was characterized, the site that is most commonly used to insert alien gene is to be arranged in the segmental thymidine kinase of genome HindIIIJ site.In some preferred vaccinia virus vector, the tk site has been processed to contain the restriction enzyme sites of 1 or 2 uniqueness, makes to use the three molecular recombination methods that prepare the library easily.Referring to preamble and Zauderer, PCT publication number WO00/028016.

The polynucleotide library of coding immunoglobulin (Ig) subunit polypeptide is inserted in poxvirus vector, the especially vaccinia virus vector, make it operably to link together with the transcription regulatory region that can in the kytoplasm of the cell of poxvirus infection, play a role.

The poxvirus transcription regulatory region comprises promotor and transcription termination signal.Genetic expression in the poxvirus is subjected to time-controllable, and the promotor of early stage, mid-term and late gene has different structures.Some poxvirus genome is the constructive expression, and the promotor of these " early-late period " genes possesses the structure of heterozygosis.Also set up synthetic morning-late promoter.Referring to HammondJ.M. etc., J.Virol.Methods66:135-8 (1997); Chakrabarti S. etc., Biotechniques23:1094-7 (1997).Among the present invention, can use any poxvirus promotor, but according to selected host cell and/or selection scheme, may be preferred early stage, late period or constitutive promoter.Usually, preferably use constitutive promoter.

The example of early promoter comprises 7.5kD promotor (also being a late promoter), DNA pol promotor, tk promotor, RNA pol promotor, 19-kD promotor, 22-kD promotor, the 42-kD promotor, 37-kD promotor, 87-kD promotor, H3 ' promotor, H6 promotor, D1 promotor, the D4 promotor, D5 promotor, D9 promotor, D12 promotor, I3 promotor, M1 promotor and N2 promotor.Referring to Moss, B. " poxvirus and duplicate " (Virology, 2d Edition, B.N.Fields, volumes such as D.M.Knipe, Raven Press, 2088 pages (1990)).The early gene identification transcription termination signal TTTTTNT that transcribes in vaccinia virus and other poxvirus, wherein N can be any Nucleotide.Transcribe usually and stop at about 50bp place, this signal upstream.Therefore, if begin expression of heterologous genes, must be noted that this signal can not be present in the coding region of these genes from the poxvirus early promoter.Referring to for example, Earl, P.L. etc., J.Virol.64:2448-51 (1990).

The example of late promoter comprises the 7.5kD promotor, MIL promotor, 37-kD promotor, 11-kD promotor, the 11L promotor, 12L promotor, 13L promotor, 15L promotor, the 17L promotor, 28-kD promotor, H1L promotor, H3L promotor, the H5L promotor, H6L promotor, H8L promotor, the D11L promotor, D12L promotor, D13L promotor, A1L promotor, A2L promotor, A3L promotor and P4b promotor.Referring to Moss, B. " poxvirus and duplicate " (Virology, 2dEdition, B.N.Fields, volumes such as D.M.Knipe, Raven Press, 2090 pages (1990)).The transcription termination signal that the obvious nonrecognition early promoter of late promoter is discerned.

Be preferred for synthetic morning-late promoter that constitutive promoter of the present invention comprises that Hammond and Chakrabarti describe, MH-5 morning-late promoter, and 7.5kD or " p7.5 " promotor.The embodiment that utilizes these promotors is disclosed in the literary composition.

Below also will go through, select and the screening method requirement causes the mechanism of necrocytosis to take place before at any cytopathic effect (CPE) that virus infection caused based on some of host cell death.The kinetics that CPE begins in the cell of infective virus depends on used virus, the type of infection multiplicity and host cell.For example, infected in the tissue culture that MOI is about 1 vaccinia virus many, CPE infects back 48 to 72 hours just obviously up to substantially exceeding.This makes 2 to 3 days time, and the CPE that not caused by carrier influences the high level expression immunoglobulin molecules, and based on antigenic selection.But, to this of some system of selection period may be not enough, when particularly using higher MOI, in addition, the time before CPE begins in purpose clone may lack.Therefore, need the virus vector that cytopathic effect weakens, especially poxvirus vector (such as vaccinia virus vector) can prolong the time period of selecting so when needed.

For example, some attenuation is realized by transgenation.They can be the mutant of complete defective, promptly need helper virus to produce infectious viral particle, and perhaps they are conditional mutants, for example the responsive to temperature type mutant.Especially the optimum condition mutant because when needs expressive host gene, can remain on the host cell of infective virus a nonpermissive environment, and for example non-permissive temperature converts the permission environment then to, and for example permissive temperature makes it to produce virion.Perhaps, can utilize the chemical inhibitor that the reversible blocking virus of specified time duplicates in infecting circulation to come complete infectious virus is carried out " attenuation ".This class chemical inhibitor includes, but are not limited to hydroxyl urea and floxuridine.When needs expressive host gene, the host cell of infective virus is maintained in the chemical inhibitor, remove chemical inhibitor then and make it to produce virion.

A large amount of attenuation poxvirus, particularly vaccinia virus have been set up.For example the bovine vaccine Ankara (MVA) of Xiu Shiing is the vaccinia virus of a plant height degree attenuation, and it derives from primary chick embryo tire inoblast to go down to posterity through 570 times and obtains (Mayr, A. etc., Infection3:6-14 (1975)).The virus that is recovered to lacked 15% wild-type bovine vaccine DNA nearly, this greatly influenced should virus host range.Reproducible or duplicating efficiency are very not low in most mammal cell lines for MVA.A peculiar property of host range restriction effect is the late phase that the blocking-up in nonpermissive cell occurs in replication cycle.The expression of late genes does not suffer damage substantially, but (Suter, G. and Moss, B.Proc Natl Acad Sci USA89:10847-51 (1992) take place to have been interrupted the form of virion; Carroll, M.W. and Moss, B.Virology238:198-211 (1997)).Even also can the synthetic virus protein of high level in non-permission host cell, this makes MVA become a safety and an expression vector efficiently especially.But, because can not finishing, MVA infects circulation in most mammalian cells, be used for more options round-robin infectious virus in order to be recovered to, need compensate the defective of MVA by its infection or superingection helper virus, this helper virus itself is a defective, can expand it and infectious MVA recombinant chou are separated by carry out difference with low MOI in the permission host cell of MVA subsequently.

Poxvirus infection has an obvious suppression effect to the protein of host cell and RNA are synthetic.These may disturb the selection that those is had the concrete poxvirus recombinant chou of specific physiological action to host cell in some cases to the influence that host gene is expressed.The vaccinia virus strain of some crucial early gene defective demonstrates host cell proteins matter synthetic restraining effect is reduced greatly.The attenuation poxvirus that lacks specific crucial early gene also has description.United States Patent (USP) 5766882 and 5770212 referring to for example Falkner etc.Can be caused the example of the crucial early gene of defective to include, but are not limited to vaccinia virus 17L, F18R, D13L, D6R, A8L, J1R, E7L, F11L, E4L, I1L, J3R, J4R, H7R and A6R gene.The crucial early gene that preferably carries out defect processing is the D4R gene of coding uracil dna glycosylase.The vaccinia virus of specific key gene generation defective is easy to breed in the compensation clone of this key gene product can be provided.

With in the text, term " compensating action " is meant that by other source such as host cell, transgenic animal or helper virus come the function of trans recovery forfeiture.The forfeiture of described function is to lose the gene product of being responsible for this function by defective virus to cause.Therefore, the defective poxvirus is the debility form of parental generation poxvirus, and can become under the situation that compensating action exists has vigor.Described host cell, transgenic animal or helper virus contain the sequence of the coding gene product of losing, or " shim member ".This shim member should be effable, can stably be incorporated in host cell, transgenic animal or the helper virus, and preferred not with or have only the genome of very little danger and defective poxvirus to recombinate.

The virus that produces in the compensation clone can infect non-compensation cell, can also high level expression early gene product.But under the situation of this key gene product not, the host closes, the packing of dna replication dna, infectious viral particle and producing can not take place.

In the text in the particularly preferred embodiment of Miao Shuing, be associated in the expressed target gene product in complicated library of coming together by expression coupling and select being structured in the vaccinia virus with the abduction delivering of shim member and target gene product.Because shim member only at those host cell inner expressions of expressing required gene product, has only these host cells can produce the infectious virus that can easily be reclaimed.

The preferred embodiment relevant with vaccinia virus can be used any poxvirus vector so that the conspicuous mode of those of ordinary skills is changed.In direct back-and-forth method, can use the carrier outside poxvirus or the vaccinia virus.

Three molecular recombination methods.Traditionally, the poxvirus vector that is out of use is identified previous unknown target gene such as vaccinia virus from complicated library, because be not used for efficient, the high titre library construction and the screening method of bovine vaccine.The ordinary method of carrying out the heterologous protein expression in vaccinia virus comprises that homologous recombination directly is connected with external in the body.Utilize homologous recombination, the recombinant virus productive rate is about 0.1% or lower.Although utilize the recombinant virus productive rate of direct connection method higher, the titre that obtains is lower.Therefore, the use of vaccinia virus vector is confined to former separated DNA is cloned to carry out protein expression and exploitation vaccine.

Three molecular recombination as disclosed in the open text WO00/028016 (Zauderer) of PCT, are a kind of new efficient, high titre production methods of cloning in vaccinia virus of being used for.Utilize three molecular recombination methods, the inventor has reached at least 90% recombinant virus productive rate, high at least 2 orders of magnitude that titre obtains than direct connection method.

Therefore, in a preferred embodiment,, in poxvirus, preferred vaccinia virus vector, make up the polynucleotide library that to express the immunoglobulin (Ig) subunit polypeptide by three molecular recombination.

" three molecular recombination " or " three molecular recombination methods " is meant a kind of like this method, this method is by with virus genomic two non-homogeneous fragments with contain the transfer vector that inserts DNA or transfer DNA importing recipient cell, allow three dna moleculars to recombinate in vivo, preparation comprises the viral genome that allos is inserted DNA thus, preferred poxvirus genome group, more preferably vaccinia virus gene group.The result of reorganization has produced the great-hearted viral genome molecule that comprises these two genomic fragments and insert DNA.Therefore, being applied to three molecular recombination methods of the present invention comprises: (a) viral genome of cutting and separating, preferred dna virus genome, more preferably linear DNA viral genome, more preferably poxvirus or vaccinia virus gene group, thereby produce first viral fragment and second viral fragment, wherein said first viral fragment and second viral fragment do not have homology; (b) provide the transferring plasmid group, these plasmids comprise the polynucleotide of coding immunoglobulin (Ig) subunit polypeptide (for example light chain immunoglobulin, heavy chain immunoglobulin or their antigen-specific fragment), it and transcription regulatory region operably link together, side joint 5 ' flanking region and 3 ' flanking region, wherein said 5 ' flanking region with (a) in the first viral fragment homology described, 3 ' flanking region with (a) in the second viral fragment homology described; And described transferring plasmid can carry out homologous recombination with first and second viral fragments, thereby forms the viral genome of living; (c) with transferring plasmid of describing in (b) and first and second viral fragments of describing (a), can carry out in the body homologous recombination (promptly at this transferring plasmid and viral fragment, three molecular recombination) import host cell under the condition, thereby produce the adorned live virus genome contain Nucleotide more than the coding immunoglobulin (Ig) subunit polypeptide; And (d) reclaim adorned viral genome by this technology preparation.Preferably, the adorned viral genome that is recovered to is packaged in the infectious viral particle.

" recombination efficiency " or " productive rate of recombinant virus " is meant in the process of preparation viral library of the present invention, the ratio of the viral total amount of recombinant virus and generation.Shown in embodiment 5, can by with the titre of recombinant virus divided by the total virus titre and multiply by 100% and calculate this efficient.For example, titre is by detecting the provirus liquid storage on suitable cell, has that under the selection situation plaque of (for example recombinant virus adds wild-type virus) is determined under (for example recombinant virus) and not selection situation.The method of selecting when especially if heterologous polynucleotide is inserted in virus thymidine kinase (tk) the site in, is well known in the art, comprises and utilizing owing to the tk gene is interrupted the resistance to bromodeoxyribouridine (BDUR) or other nucleotide analogs that causes.The example of system of selection has been described in the literary composition.

" efficient reorganization " is meant that recombination efficiency is at least 1%, and more preferably recombination efficiency about at least 2%, 2.5%, 3%, 3.5%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.

Many selective systems can be used, and include but not limited to thymidine kinase, such as herpes simplex virus thymidine kinase (Wigler etc., 1977, Cell 11:223), hypoxanthine guanine phosphoribosyltransferase (Szybalska﹠amp; Szybalski, 1962, Proc.Natl.Acad.Sci.USA48:2026) and adenine phosphoribosyltransferase (Lowy etc., 1980, Cell22:817) gene can be respectively at tk ^-, hgprt ^-, aprt ^-Use these genes in the cell.Equally, can serve as that the basis is selected following gene with the metabolic antagonist resistance: give dhfr (Wigler etc., 1980, Proc.Natl.Acad.Sci.USA77:3567 to the methotrexate resistance; O ' Hare etc., 1981, Proc.Natl.Acad.Sci.USA78:1527); Give gpt (Mulligan﹠amp to the mycophenolic acid resistance; Berg, 1981,1981, Proc.Natl.Acad.Sci.USA78:2072); Give neo to the aminoglycoside resistance (Colberre-Garapin etc., 1981, J.Mol.Biol.150:1); And give hygro to hygromycin resistance (Santerre etc., 1984, Gene30:147).

In a word, first and second viral fragments or viral genome " arm " preferably contain all virus replications and produce the necessary gene of infectious viral particle as mentioned above.Disclose the example of suitable arm in the literary composition and utilized vaccinia virus vector to produce their method.The guidance of duplicating essential zone about bovine vaccine can be with reference to the United States Patent (USP) 5770212 of Falkner etc.

But exposed poxvirus genome group DNA such as the vaccinia virus gene group, just can not produce infectious progeny when not relevant with the virion of coming in viral coded protein/function.The function of required encoding viral comprises RNA polymerase, and it can discern the bovine vaccine DNA of institute's transfection as template, initial transcribing and the duplicating of last institute's transfection DNA.United States Patent (USP) 5445953 referring to Dorner etc.

Therefore, produce infectious progeny virus in order to utilize poxvirus (such as vaccinia virus) by three molecular recombination, recipient cell preferably contains packaging function.This packaging function can be provided by helper virus, promptly with the exposed genomic dna of institute's transfection, can provide the virus of duplicating and assembling the necessary suitable protein and the factor of progeny virus.

Helper virus can be closely-related virus, for example belongs to the poxvirus of identical subfamily with bovine vaccine, can be from identical or different genus.In this situation, it is useful that selection can provide the helper virus of RNA polymerase, and described RNA polymerase can be discerned the DNA of institute's transfection as template, initial transcribing and the duplicating of last institute's transfection DNA.If with very relevant virus as helper virus, thereby be useful to the formation that it carries out attenuation infringement infectious virus.For example, can be non-allowable temperature use temperature responsive type helper virus.Preferably, use the allos helper virus.This class example includes, but are not limited to fowlpox virus (avipox) such as fowlpox virus (fowlpox), perhaps lacks acropathy poison (mousepox) virus.Preferred especially fowlpox virus because they can provide essential subsidiary function, but can not duplicate or produce infectious virion (Scheiflinger etc., Proc.Natl.Acad.Sci.USA89:9977-9981 (1992)) in mammalian cell.Use allos virus can reduce helper virus genome and institute and infects reorganization between the genome, this is the meeting generation when being binned in the homologous sequence that has very close virus in the cell.Referring to Fenner﹠amp; Comben, Virology5:530 (1958); Fenner, Virology8:499 (1959).

Perhaps, can be by genetic elements but not helper virus provides the essential subsidiary function of recipient cell.For example, can come composition ground to produce subsidiary function by transformed host cell, perhaps with the temporary transient transfection host cell of plasmid of expressing subsidiary function, with the retroviral infection host cell of expressing subsidiary function, perhaps provide by any other expression vector that is fit to the required helper viral function of expression.United States Patent (USP) 5445953 referring to Corner etc.

According to three molecular recombination methods, the first and second viral genome fragments can not interconnect or recombinate, and promptly they do not contain compatible sticking end or homology zone, and perhaps, sticking end was handled by the dephosphorylation enzyme.In preferred embodiments, viral genome comprises first recognition site of first restriction enzyme and second recognition site of second restriction enzyme, described first and second viral fragments are to produce by the digestion with restriction enzyme viral genome with suitable generation virus " arm ", and separate this first and second viral fragment by ordinary method.Ideal situation is, the first and second restriction enzyme enzyme recognition sites are unique in viral genome, perhaps, cut the virus " arm " that produces the gene comprise all key functions with two kinds of restriction enzymes, promptly first and second recognition sites physics in viral genome is arranged to infectious optional to virus of the zone that extends between first and second viral fragments.

At one vaccinia virus vector is used for the preferred embodiment of three molecular recombination methods, has used and contain the virus genomic vaccinia virus vector that two unique restriction sites are arranged in the tk gene.In some preferred vaccinia virus gene group, described first restriction enzyme is NotI, and its recognition site is the GCGGCCGC in the tk gene, and second restriction enzyme is ApaI, and its recognition site is the GGGCCC in the tk gene.More preferably comprise the virus genomic vaccinia virus vector of v7.5/tk viral genome or vEL/tk.

According to this embodiment, used such transferring plasmid, it have can with the vaccinia virus gene group in contain thymidine kinase gene the zone carry out the flanking region of homologous recombination.Can use easily and contain the segmental fragment of HindIII-J in the vaccinia virus gene group, described HindIII-J fragment contains the tk gene.

When described virus vector was poxvirus, the preferred polynucleotide that insert had operably connected the pox viruses express regulating and controlling sequence, have more preferably connected strong composing type poxvirus promotor, such as p7.5 or in early days synthetic/late promoter.

Correspondingly, transferring plasmid of the present invention comprises coding immunoglobulin (Ig) subunit polypeptide (for example heavy chain and light chain immunoglobulin, the perhaps antigen-specific fragment of heavy chain or light chain) polynucleotide, it has operably connected poxvirus p7.5 promotor, perhaps synthetic early stage/late promoter.

Preferred transferring plasmid of the present invention is pVHE, and it contains the polynucleotide of the coding heavy chain immunoglobulin polypeptide that has operably connected poxvirus p7.5 promotor, and it comprises sequence:

GGCCAAAAATTGAAAAACTAGATCTATTTATTGCACGCGGCCGCAAACCATGGGATGGAGCTG

TATCATCCTCTTCTTGGTAGCAACAGCTACAGCCCGCATATGGTCCACCGTCTCCTCAGGGAG

TGCATCCGCCCCAACCCTTTTCCCCCTCGTCTCCTGTGAGAATTCCCCGTCGGATACGAGCAG

CGTGGCCGTTGGCTGCCTCGCACAGGACTTCCTTCCCGACTCCATCACTTTCTCCTGGAAATA

CAAGAACAACTCTGACATCAGCAGCACCCGGGGCTTCCCATCAGTCCTGAGAGGGGGCAAGTA

CGCAGCCACCTCACAGGTGCTGCTGCCTTCCAAGGACGTCATGCAGGGCACAGACGAACACGT

GGTGTGCAAAGTCCAGCACCCCAACGGCAACAAAGAAAAGAACGTGCCTCTTCCAGTGATTGC

TGAGCTGCCTCCCAAAGTGAGCGTCTTCGTCCCACCCCGCGACGGCTTCTTCGGCAACCCCCG

CAGCAAGTCCAAGCTCATCTGCCAGGCCACGGGTTTCAGTCCCCGGCAGATTCAGGTGTCCTG

GCTGCGCGAGGGGAAGCAGGTGGGGTCTGGCGTCACCACGGACCAGGTGCAGGCTGAGGCCAA

AGAGTCTGGGCCCACGACCTACAAGGTGACTAGCACACTGACCATCAAAGAGAGCGACTGGCT

CAGCCAGAGCATGTTCACCTGCCGCGTGGATCACAGGGGCCTGACCTTCCAGCAGAATGCGTC

CTCCATGTGTGTCCCCGATCAAGACACAGCCATCCGGGTCTTCGCCATCCCCCCATCCTTTGC

CAGCATCTTCCTCACCAAGTCCACCAAGTTGACCTGCCTGGTCACAGACCTGACCACCTATGA

CAGCGTGACCATCTCCTGGACCCGCCAGAATGGCGAAGCTGTGAAAACCCACACCAACATCTC

CGAGAGCCACCCCAATGCCACTTTCAGCGCCGTGGGTGAGGCCAGCATCTGCGAGGATGACTG

GAATTCCGGGGAGAGGTTCACGTGCACCGTGACCCACACAGACCTGCCCTCGCCACTGAAGCA

GACCATCTCCCGGCCCAAGGGGGTGGCCCTGCACAGGCCCGATGTCTACTTGCTGCCACCAGC

CCGGGAGCAGCTGAACCTGCGGGAGTCGGCCACCATCACGTGCCTGGTGACGGGCTTCTCTCC

CGCGGACGTCTTCGTGCAGTGGATGCAGAGGGGGCAGCCCTTGTCCCCGGAGAAGTATGTGAC

CAGCGCCCCAATGCCTGAGCCCCAGGCCCCAGGCCGGTACTTCGCCCACAGCATCCTGACCGT

GTCCGAAGAGGAATGGAACACGGGGGAGACCTACACCTGCGTGGTGGCCCATGAGGCCCTGCC

CAACAGGGTCACTGAGAGGACCGTGGACAAGTCCACCGAGGGGGAGGTGAGCGCCGACGAGGA

GGGCTTTGAGAACCTGTGGGCCACCGCCTCCACCTTCATCGTCCTCTTCCTCCTGAGCCTCTT

CTACAGTACCACCGTCACCTTGTTCAAGGTGAAATGAGTCGAC

Called after SEQ ID NO:14 in the literary composition.The variable region of heavy chain of pcr amplification can be met the BssHII (the Nucleotide 96-100 of SEQ ID NO:15) and BstEII (the Nucleotide 106-112 of SEQ ID NO:16) the single endonuclease digestion site that show with boldface letter in the frame ground insertion sequence.

In addition, pVHE can be used for some embodiments like this, promptly needs as mentioned above to transfer in the plasmid vector so that select the polynucleotide in second library subsequently from the polynucleotide in first library separating.

Another preferred transferring plasmid of the present invention is pVKE, and it comprises the polynucleotide of the coding immunoglobulin (Ig) kappa light chain polypeptide that has operably connected poxvirus p7.5 promotor, and it contains sequence:

GGCCAAAAATTGAAAAACTAGATCTATTTATTGCACGCGGCCGCCCATGGGATGGAGCTGTAT

CATCCTCTTCTTGGTAGCAACAGCTACAGGCGTGCACTTGACTCGAGATCAAACGAACTGTGG

CTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTG

TTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG

CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACA

GCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCG

AAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGG

TCGAC

Called after SEQ ID NO:17 in the literary composition.The kappa variable region of light chain of pcr amplification can be met the ApaLI (the Nucleotide 95-100 of SEQ ID NO:18) and XhoI (the Nucleotide 105-110 of SEQ ID NO:19) the single endonuclease digestion site that show with boldface letter in the frame ground insertion sequence.

In addition, pVKE can be used for some embodiments like this, promptly need in the process of the polynucleotide of selecting first library polynucleotide that are present in second library in the plasmid vector be arranged as mentioned above.

Another preferred transferring plasmid of the present invention is pVLE, and it comprises the polynucleotide of the coding immunoglobulin (Ig) lambda light chain polypeptide that has operably connected poxvirus p7.5 promotor, and it contains sequence:

GGCCAAAAATTGAAAAACTAGATCTATTTATTGCACGCGGCCGCCCATGGGATGGAGCTGTAT

CATCCTCTTCTTGGTAGCAACAGCTACAGGCGTGCACTTGACTCGAGAAGCTTACCGTCCTAC

GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA

CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGG

TGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACA

GCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCT

ACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAG

AGTGTTAGGTCGAC

Called after SEQ ID NO:20 in the literary composition.The lambda light chain variable district of pcr amplification can be met the ApaLI (the Nucleotide 95-100 of SEQ ID NO:21) and HindIII (the Nucleotide 111-116 of SEQ ID NO:22) the single endonuclease digestion site that show with boldface letter in the frame ground insertion sequence.

In addition, pVlE can be used for some embodiments like this, promptly need in the process of the polynucleotide of selecting first library polynucleotide that are present in second library in the plasmid vector be arranged as mentioned above.

" insert DNA " and be meant one or more allogeneic dna sequence DNA fragment that in the recombinant virus carrier, to express.According to the present invention, " inserting DNA " is the polynucleotide of coding immunoglobulin (Ig) subunit polypeptide.Described DNA section can be natural, non-natural, synthetic or their combination.The method for preparing insertion DNA of the present invention is disclosed in the literary composition.

" transferring plasmid " is meant and contains the plasmid vector that is positioned at the insertion DNA between 5 ' flanking region and the 3 ' flanking region as mentioned above.This 5 ' flanking region and first viral fragment have homology, and the 3 ' flanking region and second viral fragment have homology.Preferably, described transferring plasmid contains suitable promotor, when being poxvirus such as virus vector, contains strong composing type bovine vaccine promotor at the insertion dna upstream.Term " carrier " is meant the polynucleotide constructs that contains the heterologous polynucleotide section, and it can be with described polynucleotide sector transfer to proper host cell.The polynucleotide that preferably are contained in the carrier have operably connected the suitable regulating and controlling sequence that can cause that polynucleotide are expressed in appropriate host.This class regulating and controlling sequence comprises and causes the promotor of transcribing, the optional operon sequence of regulatory transcription, the sequence of the suitable mRNA ribosome bind site of encoding, and the sequence of regulatory transcription and translation termination.With in the present invention, carrier can be a plasmid, phage particle, and virus, messenger RNA(mRNA), perhaps just a potential genome inserts fragment.In case be transformed in the appropriate host, described carrier can duplicate and be independent of host genome and play a role, and perhaps in some cases, is incorporated in the genome itself.The typical plasmid expression vector that is used for the mammalian cell culture expression for example is based upon pRK5 (EP307247), on the basis of pSV16B (WO91/08291) and pVL1392 (Pharmingen).

But " transferring plasmid " that use in the literary composition is not limited to specific plasmid or carrier.The DNA section of any annular or linear or other suitable forms can be transferred to the interior carrier of host cell with first and second viruses " arm " as in the three molecular recombination methods described DNA being inserted fragment.Other suitable carriers comprise lambda particles phage that describe in the literary composition or known in the art, mRNA, dna fragmentation etc.Many plasmids can be " the elementary libraries " such as the lambda particles phage of describing in the literary composition.

The improvement of three molecular recombination.Three molecular recombination can be used for making up titre in vaccinia virus and be approximately 10 ⁷The cDNA library of the pfu order of magnitude.The diversity in several factors limit has been arranged this class cDNA library or other libraries.They comprise: the size in elementary cDNA library or other libraries, such as the polynucleotide library that can be structured in the coding immunoglobulin (Ig) subunit polypeptide in the plasmid vector, and purifying a large amount of (several hectogamma) virus " arm " (preferred vaccinia virus " arm " or other poxvirus " the arm ") work of paying.Improve three molecular recombination so that bovine vaccine or other viral DNAs can with elementary cDNA library or other libraries (such as be structured in lambda particles phage or by the polynucleotide of the coding immunoglobulin (Ig) subunit polypeptide in its deutero-DNA or the phagemid) recombinate, perhaps make and behind the virus vector that has infected modified, in body, to prepare independent viral DNA arm, so just can improve greatly with the eucaryon virus cDNA library of this method structure or the quality and the titre in other libraries.

CDNA is inserted fragment transfer to vaccinia virus from the lambda particles phage library.λShi Juntizaiti has some advantages in the construction cDNA library or aspect other libraries such as polynucleotide of coding immunoglobulin (Ig) subunit polypeptide than plasmid vector.Plasmid cDNA (or other DNA insert fragment) library or linear DNA library can transform or electroporation importing bacterial cell by chemistry/thermal shock is frightened.The plasmid that the preferred transformation ratio of bacterial cell is less so just causes losing longer cDNA or other insertions DNA in the library, such as the polynucleotide of coding immunoglobulin (Ig) subunit polypeptide.In addition, conversion is the lower process of efficiency ratio with foreign DNA or other DNA transfered cells, for construction cDNA library or the expensive commercialization competence bacterium of other libraries (such as the polynucleotide of coding immunoglobulin (Ig) subunit polypeptide) needs use.On the contrary, λShi Juntizaiti can tolerate that 12kb or longer cDNA insert fragment, and without any big or small skewed popularity.Utilize efficiently that the commercial package extract can become virion with the λ carrier package external, can recombinant chou λ genome be imported bacterial cell by infecting then.What this process produced and obtained in plasmid library usually compares, and has higher titre and reflects that better big cDNA or other insert the elementary library of DNA (such as the polynucleotide of the immunoglobulin (Ig) subunit polypeptide of encoding).

In order to insert fragment or other DNA insertion fragment from the cDNA that is structured in the λ carrier, polynucleotide such as coding immunoglobulin (Ig) subunit polypeptide, transfer to the eucaryon virus vector such as in the vaccinia virus, the λ carrier modification can must be carried out homologous recombination with vaccinia virus DNA thereby make it to comprise the vaccinia virus dna sequence dna.The following examples have been used the vaccinia virus homologous sequence, but can use other virus similarly.For example, can cut the vaccinia virus HindIII J fragment (containing bovine vaccine tk gene) (the bovine vaccine dna sequence dna of 3kb) that plasmid p7.5/ATGO/tk (as described in following examples 5) is comprised with HindIII and SnaBI, subclone forms pT7B3.Vtk to the HindIII/SnaBI site among the pT7Blue3 (Novagen article No. 70025-3).Can from this carrier, downcut bovine vaccine tk gene and insert SacI/SmaI site among the LambdaZap Express (Stratagene) with SacI and SnaBI, thereby produce Lambda.Vtk.This Lambda.Vtk carrier contains NotI, BamHI, and SmaI and SalI single endonuclease digestion site are used in bovine vaccine 7.5k promotor downstream insertion cDNA. and can adopt method well known in the art construction cDNA library in Lambda.Vtk.

Can adopt from the DNA that is structured in cDNA library in Lambda.Vtk or any similar phage or other libraries (such as the polynucleotide of coding immunoglobulin (Ig) subunit polypeptide) to prepare cDNA or other insert DNA recombinant chou vaccinia virus, described DNA comprises that the cDNA that has the flank bovine vaccine dna sequence dna that is used for promoting homologous recombination inserts fragment or other insert DNA.The method of downcutting plasmid by coinfection helper phage (ExAssist phage, Stratagene article No. 211203) from the λ genome is well known in the art.From being equal to cDNA library or other libraries in the plasmid vector based on cutting on a large scale the library of λ to have produced to be present in.The plasmid that scales off from Lambda.Vtk cDNA library for example contains bovine vaccine tk sequence, and side is that cDNA inserts fragment or other insert DNA, such as the polynucleotide of coding immunoglobulin (Ig) subunit polypeptide.This plasmid DNA can be used for making up the bovine vaccine recombinant chou by three molecular recombination then.Another embodiment of this method is direct purifying λ DNA from primary Lambda.Vtk library, and this recombinant virus (λ) DNA or its fragment are carried out three molecular recombination with two big vaccinia virus dna fragmentation transfections.

Produce the bovine vaccine arm in the body.The purifying of bovine vaccine DNA or other viral DNAs " arm " and transfection are limiting factors that makes up the polynucleotide library by three molecular recombination.This method improved make and to produce viral arm in vivo that especially the vaccinia virus arm just can more effectively make up the library in eucaryon virus.

Can modify host cell makes its expression can discern the restriction enzyme that imports to genomic certain the unique site of virus vector.For example, during these host cells of vaccinia virus infection, described restriction enzyme will digest bovine vaccine DNA, and generation can only be repaired " arm " of (promptly reconnecting) by three molecular recombination.The example of restriction enzyme comprises bacterial enzyme NotI and ApaI, yeast restriction endonuclease VDE (R.Hirata, Y.Ohsumi, A.Nakano, H.Kawasaki, K.Suzuki, Y.Anraku.1990 J.Biological Chemistry265:6726-6733), Chlamydomonas eugametos restriction endonuclease I-CeuI and other restriction endonucleases well known in the art.For example, make up the bovine vaccine strain that its tk gene contains NotI and ApaI single endonuclease digestion site, can make up the virus strain that contains VDE and/or I-CeuI single endonuclease digestion site in the tk gene by means commonly known in the art at an easy rate.

Constitutive expression restriction enzyme pair cell is lethal, because this enzyme can be with chromosomal dna fragmentization.For fear of this stubborn problem, make it under the inducible promoter regulation and control, express restriction endonuclease gene the host cell modification in one embodiment.

A preferred inducible expression method has been utilized Tet-On Gene ExpressionSystem (Clontech).In this system, the expression of gene of coding restriction endonuclease is reticent under the situation that lacks inductor (tsiklomitsin).This just feasible can being separated to can be by the stable transfectional cell series of abduction delivering virulent gene (being restriction endonuclease) (Gossen, M. etc., Science268:1766-1769 (1995)).Add the tetracycline derivant doxycycline and can induce the expression of restriction endonuclease.In a preferred embodiment, with the Tet-On carrier of regulation and control NotI genetic expression transfection BSC1 host cell stably.Induce the monolayer that is paved with doxycycline, infect v7.5/tk (NotI single endonuclease digestion site is arranged in the tk gene) then, and with cDNA or insert DNA recombinant chou transferring plasmid or transfer DNA or lambda particles phage or phagemid dna carry out transfection.The NotI single endonuclease digestion site in tk gene or other sequences for example among the bovine vaccine DNA that the Not I restriction endonuclease digestion of host cell coding exposes; thereby produce two big bovine vaccine dna fragmentations, these two fragments have only and carry out three molecular recombination with transferring plasmid or phage DNA and could form the total length viral DNA.Estimate to stop the generation of adorned infectious virus with NotI digestion host cell chromosome DNA, because in the process of virus replication and virion assembling, do not need host cell to breed.

In with this method body, prepare in another embodiment of viral arm (such as the bovine vaccine arm), made up the bovine vaccine strain of a modification, contain unique restriction enzyme site in its tk gene or other dispensable genes, and the heterologous polynucleotide that is in the coding restriction endonuclease under the regulation and control of T7 phage promoter is contained in another nonessential site in the bovine vaccine genome.To cause the expression of restriction endonuclease to the infection of the cell of expressing t7 rna polymerase, and this enzymic digestion bovine vaccine DNA subsequently.In a preferred embodiment, by at HindIIIC or F district (Coupar, E.H.B. etc., Gene68:1-10 (1988); Flexner, C. etc., Nature330:259-262 (1987)) in insert its expression and be subjected to the encode box of cDNA of NotI of containing of T7 promoter regulation, strain is modified to bovine vaccine v7.5/tk, thus formation v7.5/tk/T7NotI.With the cDNA that is in the coding T7 RNA polymerase under the mammalian promoter regulation and control stably transfectional cell series (O.Elroy-Stein, B.Moss.1990Proc.Natl.Acad.Sci.USA87:6743-6747).This package cell line can cause the T7 RNA polymerase dependency of NotI to express after infecting v7.5/tk/T7NotI, and subsequently bovine vaccine DNA is digested to arm.Infectious total length viral DNA can only be reformulated and be packaged as to the bovine vaccine DNA arm that is digested after carrying out three molecular recombination with transferring plasmid or phage DNA.In another embodiment of this method, can pass through coinfection T7 RNA polymerase recombinant chou helper virus, provide T7 RNA polymerase (P.Britton such as fowlpox virus, P.Green, S.Kottier, K.L.Mawditt, Z.Penzes, D.Cavanagh, M.A.Skinner.1996 J.General Virology77:963-967).

The digestion that characteristics using three molecular recombination of the big viral dna fragment (preferred bovine vaccine dna fragmentation) of the interior generation of these Different Strategies bodies are bovine vaccine DNA can be carried out before reorganization, but and nonessential like this.It has guaranteed to have only recombinant virus can escape the destruction that digestion causes.These three molecular recombination with the bovine vaccine DNA that adopts the transfection external digestion form contrast, must prepare the bovine vaccine dna fragmentation in the latter before reorganization.Carrying out frequency ratio that two molecular recombination obtain recombinant chou before digestion, to obtain the frequency of recombinant chou higher by three molecular recombination after digestion be possible.

Utilize virus vector, especially poxvirus separates the selection and the screening strategy of recombinant chou immunoglobulin molecules.In certain embodiments of the invention, utilized three molecular recombination methods to produce the polynucleotide library of expressing the immunoglobulin (Ig) subunit polypeptide.In this embodiment, comprising total length immunoglobulin (Ig) subunit polypeptide or its segmental library is box preparation by at first inserting coding constant region for immunoglobulin and signal peptide in the transferring plasmid that contains with vaccinia virus homologous 5 ' and 3 ' district.The immune globulin variable region of resetting separates from the B cell of the pre B cell of nonimmune animal or immune animal or plasmocyte by PCR.These PCR fragment clonings between immunoglobulin (Ig) signal peptide and the constant region, and are conformed to their frames, thus the coding region that produces the many skins of described immunoglobulin (Ig) subunit.These transferring plasmids are imported the host cell that poxvirus " arm " arranged, utilize three molecular recombination methods to produce the library.

The invention provides multiple evaluation (promptly selecting or screening) and have the method for required specific immunoglobulin molecules, wherein said immunoglobulin molecules is external generation in eukaryotic cell.These methods comprise selects the host cell effect, such as the necrocytosis of antigen induction and the signal transmission of antigen induction; Antigen-specific among the screening host cell group screens to see whether there is the solubility immunoglobulin molecules with required antigen-specific or functional characteristics in conjunction with situation and to the substratum that the host cell group is grown.

As what describe in detail in the literary composition, the invention provides necrocytosis according to antigen induction, the signal transmission of antigen induction, antigen-specific combination or other antigen-specific sexual functions are identified immunoglobulin molecules or the segmental method of expressing in the eukaryotic cell of its antigen-specific.Selection of the present invention and triage techniques have been eliminated skewed popularity that selection antibody brings in rodent or the limitation of synthesizing and assembling in bacterium.

Many authentication methods of describing in the literary composition depend on the expression of host cell gene or host cell transcription regulatory region, they in response to antigen be expressed in host cell surface on immunoglobulin molecules or segmental combination of its antigen-specific and inducing cell death or produce can detected signal directly or indirectly.Should be mentioned that the most preferred embodiment of the present invention need use the eucaryon virus vector, preferred poxvirus vector, more preferably vaccinia virus vector comes host cells infected.Those of ordinary skills are readily appreciated that in some clone, in case infect poxvirus, even without the expression of virogene, some protein synthesis of host cell also can be closed rapidly.If need to raise host cell gene or host cell transcription regulatory region for the necrocytosis of inducing antigen inductive or cell signal transmission, this will have problems.But this problem is not insurmountable, because in some clone, before viral dna replication, it is incomplete that the host protein synthetic is suppressed.Referring to Moss, B. " Poxviridae and duplicate " (Virology, 2 ^NdEdition, B.N.Fields, volumes such as D.M.Knipe, Raven Press, 2096 pages (1990)).But this need promptly screen a large amount of host cells and see that they are at infection eucaryon virus vector (preferred poxvirus vector, more preferably vaccinia virus vector) time, expresses those and ability of the gene product that raised when crosslinked takes place at immunoglobulin molecules with surface expression; Also to screen and see that can they the differential expression cytogene when infecting various mutant and attenuated virus target host cell.

Correspondingly, the invention provides a kind of express spectra of particular host cell on the little battle array in orderly cDNA library that utilize, a large amount of host cells are screened see that they are when the infective virus carrier, the steering quality of the expression of host cell gene and/or host cell transcription regulatory region, these have influence on the necrocytosis or the cell signal transmission of antigen induction.Duggan, (Nature Genet.21 (1 Suppl): 10-14 (1999)) described express spectra in little battle array, this article hereby incorporated by reference such as D.J..

According to this method, utilize more not host cells infected and infected eucaryon virus expression carrier (preferred poxvirus vector of express spectra, the gene expression pattern of host cell more preferably vaccinia virus vector), wherein this concrete eucaryon virus vector is exactly the carrier that is used to make up the first and second polynucleotide libraries of the present invention.Like this, just can identify in its surface expression immunoglobulin molecules or its antigen-specific fragment, and further when infecting given virus, express the proteinic proper host cell of required inducibility.

Express spectra also can be used for the host cell gene expression pattern in the more given host cell, the expression pattern of the host cell when for example relatively having infected the host cell of complete venereal infection poisonous carrier and having infected its corresponding attenuated virus carrier.Express spectra in microarray makes and can the host cell that infect multiple attenuated virus be screened on a large scale that wherein attenuation is realized by various different modes.For example, some attenuation is realized by transgenation.Many vaccinia mutant bodies have obtained research.These can be the mutant of complete defective, and promptly producing infectious viral particle needs helper virus, and perhaps they may be conditional mutants, for example the responsive to temperature type mutant.Especially the optimum condition mutant because when needs expressive host gene, can remain on the host cell of virus infection non-permission environment (for example remaining on non-allowable temperature), changes into then to allow environment (for example allowable temperature) so that produce virion.Perhaps, can utilize infect round-robin determine point reversibly the chemical inhibitor that duplicates of blocking virus come complete infectious virus " attenuation ".Described chemical inhibitor includes, but are not limited to hydroxyl urea and floxuridine.When needs expressive host gene, the host cell of infective virus is maintained in the chemical inhibitor, remove chemical inhibitor then and make it to produce virion.

Utilize this method, identify proper host cell, suitable transcription regulatory region, and/or suitable attenuated virus in any system of selection that the express spectra in little battle array can be used for describing in the text.

In one embodiment, provide a kind of selection coding immunoglobulin molecules or the segmental method of its antigen-specific, this method is based on the apoptosis of direct antigen induction.According to this method, selection is used to infect and/or the host cell of transfection is early stage B cell lymphoma.Suitable early stage B cell lymphoma clone comprises, but be not limited to the CH33 cell, CH31 cell (Pennell, C.A. etc., Proc.Natl.Acad.Sci.USA82:3799-3803 (1985)), or WEHI-231 cell (Boyd, A.W. and Schrader, J.W.J.Immunol.126:2466-2469 (1981)).Early stage B cell lymphoma clone is replied (Pennell, C.A. and Scott, D.W.Eur.J.Immunol.16:1577-1581 (1986) by inducing the death of spontaneous growth-inhibiting and programmed cellization to the crosslinked generation of antigen specific immunoglobulin; Tisch, R. etc., Proc.Natl.Acad.Sci.USA85:69114-6918 (1988); Ales-Martinez, J.E. etc., Proc.Natl.Acad.Sci.USA85:69119-6923 (1988); Warner, G.L. and Scott, D.W.Cell.Immunol.115:195-203 (1988)).Infect as described above and/or the transfection first and second polynucleotide libraries after, synthetic and the assembling of antibody molecule was carried out about 5 to 48 hours, preferably approximately 6 hours, about 10 hours, about 12 hours, about 16 to 20 hours, about 24 to 30 hours, about 36 hours, about 40 hours, perhaps about 48 hours, more preferably about 12 hours or about 24 hours; During this period of time the host is contacted with specific antigen, so that crosslinked any specific immunoglobulin receptor (is membrane-bound immunoglobulin molecules, or its antigen-specific fragment), and in those express the host cell of immunoglobulin (Ig)s cell death inducing, described host cell suppresses by induced growth and programmed cell death and the crosslinked generation of the former specific immunoglobulin of facedown is replied.Reclaim apoptotic host cell takes place, perhaps their content, the polynucleotide that comprise the coding immunoglobulin (Ig) subunit polypeptide that wherein comprises, the first library polynucleotide of the first immunoglobulin (Ig) subunit polypeptide thereby enrichment is encoded, the described first immunoglobulin (Ig) subunit polypeptide is as immunoglobulin molecules, or the segmental part of its antigen-specific, binding purposes antigen specifically.

Polynucleotide in further selection and enrichment first library, and after separating these polynucleotide, process reclaims the polynucleotide in second library like the implementation of class, and they are as immunoglobulin molecules, or the segmental part of its antigen-specific, can be in conjunction with specific purpose antigen.

Fig. 1 has shown an example of this method.At poxvirus vector, make up in the preferred vaccinia virus vector in " first library ", polynucleotide encoding wherein is not from by the multiple heavy chain of the antibody produced cell of immunity or immune donor; In plasmid vector, make up the similar variation " second library " of the polynucleotide of coding light chain immunoglobulin, wherein the expression of polynucleotide is subjected to the bovine vaccine promotor, preferred synthetic is early stage/late promoter, and the regulation and control of for example p11 promotor, or p7.5 promotor.For this embodiment, preferably that the poxvirus construct is coded immunoglobulin heavy chain constant region is designed to keep strides the film district, thereby immunoglobulin receptor is expressed on the surface film.With infection multiplicity is the poxvirus heavy chain library infection of eukaryotic cells of about 1 (MOI=1), preferred early stage B cell lymphoma cell.After 2 hours, with light chain plasmid library transfection infected cells under the condition that allows each cell on average to absorb 10 or more a plurality of independent light chain plasmid recombinant and make it to express.Because the expression of recombinant gene is subjected to the regulation and control of vaccinia virus promotor in this plasmid, do not need to take place nuclear and integrate, just can be in the kytoplasm of the cell that has infected vaccinia virus the high level expression recombinant gene product.In addition, annular DNA does not rely on the transfection light chain plasmid recombinant (Merchlinsky, M. and Moss, B.Cancer Cell 6:87-93 (1988)) that mechanism that sequence increases will produce greater concn in the kytoplasm of the cell that has infected vaccinia virus.These two factors cause high level expression, cause that excessive light chain is synthetic.

Another preferred embodiment has utilized in the cell of expressing the T7 RNA polymerase activated T7 phage promoter to regulate and control the expression of polynucleotide, described polynucleotide encoding is structured in poxvirus vector, " first library " in the preferred vaccinia virus vector, polynucleotide encoding wherein is from the multiple heavy chain of the antibody produced cell of not immune or immune donor; And be structured in the plasmid vector similar variation " second library " (the Eckert D. and Merchlinsky M.J Gen Virol.80 (Pt6): the 1463-9 (1999) of the polynucleotide of coding light chain immunoglobulin; Elroy-Stein O., Fuerst T.R. and MossB.Proc.Natl.Acad.Sci.USA86 (16): 6126-30 (1989); FuerstT.R., Earl P.L. and Moss B.Mol Cell Biol.7 (7): 2538-44 (1987); Elroy-Stein O. and Moss B.Proc.Natl.Acad.Sci.USA87 (17): 6743-7 (1990); Cottet S. and Corthesy B.Eur J Biochem246 (1): 23-31).

Those of ordinary skills are readily appreciated that, consideration aspect design it is important kinetics during this experiment, because the expression vector derived from poxvirus self is cytopathogenic in about 1 to 10 day time period, this time period is more frequent to be about 2 to 8 days, 2 to 6 days, perhaps 2 to 4 days, depend on used virus vector, concrete host cell and infection multiplicity.In a preferred embodiment, select B cell lymphoma, wherein the apoptosis reaction to the crosslinked generation of surface immumoglobulin is rapider than the natural cytopathic effect of poxvirus infection in this cell.Therefore, after the apoptosis that the immunoglobulin molecules on preferred host cell surface causes owing to antigen induction sexual intercourse connection occurs in host cell and antigen contacts about 1 hour to 4 days is so that generation before the inducing of CPE.More preferably, after apoptosis occurs in host cell and antigen contacts about 1 hour, 2 hours, about 3 hours, 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 28 hours, about 32 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours.More preferably contacting the about 12 hours inner cell apoptosis in back with antigen at host cell is induced.That perhaps, can adopt cytopathic effect induces the slower attenuation poxvirus of kinetics.The poxvirus vector of attenuation is disclosed in the literary composition.

According to this method, when apoptosis takes place, select the host cell of its surface expression antigen specific immune sphaeroprotein.For example, if host cell attached on the solid substrate, apoptotic cell takes place and will discharge from matrix in those, the liquid nutrient medium of cultivating host cell by results reclaims them.Perhaps, host cell is attached on the solid substrate, and apoptotic cell takes place for those the hydrolysis phenomenon can occur, thereby its kytoplasm content is discharged in the liquid nutrient medium of cultivating host cell.Can from liquid nutrient medium, gather in the crops the virion that these cells discharge then.

The host cell that contains the polynucleotide of coding immunoglobulin (Ig) subunit polypeptide may be because certain mechanism becomes " not adherent " or " debility ", this class mechanism comprises hydrolysis, can not be adherent, lose vitality, lose the integrity of film, lose structural stability, the cytoskeleton composition is destroyed, can not keep membrane potential, cell cycle arrest, can not generate energy etc.Therefore can pass through any physical means, such as sucking-off, washing is filtered, and centrifugal, cell sorting, fluorescence-activated cell sorting (FACS) wait and reclaim the host cell that contains polynucleotide of interest, promptly open with all the other cellular segregation.

For example, cracking may take place in the host cell that contains the polynucleotide of coding immunoglobulin (Ig) subunit polypeptide, therefore recombinant virus particle (preferred poxvirus particle, more preferably vaccinia virus particle) is discharged in the substratum, perhaps become not adherent, therefore break away from solid support.So, in the preferred embodiment, the recombinant virus of release and/or not adherent cell and attached cell are separated by sucking-off or washing.

Host cell is in the situation of early stage B cell lymphoma clone, can make these cells by being attached on the solid substrate with the interaction that is combined in the B cell-specific antibody on the matrix.Suitable B cell-specific antibody includes, but are not limited to anti-CD 19 antibodies and anti-CD20 antibodies.

In other preferred embodiments, utilize transfection the host cell of construct cause the necrocytosis of antigen induction directly or indirectly, external polynucleotide in the described construct (it is expressed and causes necrocytosis indirectly) have operably connected transcription regulatory region, and the latter is induced when surface immumoglobulin is molecule crosslinked.

" derivative transcription regulatory region when surface immumoglobulin is molecule crosslinked " is meant a kind of like this district, host cell promotor for example, and it regulates and control the gene that those immunoglobulin molecules at surface expression are raised when crosslinked usually.A preferred example of this class transcription regulatory region is the BAX promotor, and it is raised when B cell lymphoma cell generation surface immumoglobulin is molecule crosslinked in early days.

(shown in Fig. 2 A and 2B) in one embodiment, the method for inducing cell death when providing a kind of external polynucleotide to be expressed in Codocyte toxicity T cell (CTL) epi-position.The external polynucleotide of described coding CTL epi-position are placed in transcription regulatory region and operably link together, and this control region is induced when surface immumoglobulin is molecule crosslinked.When the immunoglobulin molecules generation antigen induction of host cell surface crosslinked, the CTL epi-position is expressed on the host cell surface, is in the MHC molecular range of determining that also is expressed in host cell surface.Cell is contacted with discerning the epitope specificity CTL that is in the CTL epi-position in definite MHC molecular range, and the cracking phenomenon takes place rapidly in the cell of expressing the CTL epi-position.The method of selecting and reclaim the host cell of expressing special CTL epi-position is further disclosed in the open text WO00/028016 of the PCT of Zauderer.

Selecting host cell is to finish by reclaiming those cell or its contents that necrocytosiss take place and/or the cracking phenomenon occurs.For example, if selected the host cell of apposition growth on solid support, then those host cells that necrocytosiss take place and/or the cracking phenomenon occurs will discharge from upholder, are recovered in cell conditioned medium liquid.Perhaps, can from cell conditioned medium liquid, be recovered to the virion that host cell discharged that those necrocytosis take place and/or the cracking phenomenon occurs.

According to this embodiment, the expressed MHC molecule of host cell surface can be I class MHC molecule or II class MHC molecule.The MHC molecule of expressing on the host cell in particularly preferred embodiments, is H-2K ^dMolecule, the CTL epi-position of being expressed when antigen induction crosslinked takes place is peptide GYKAGMIHI, called after SEQ ID NO:23 in the literary composition.

When utilizing this method, can use can be at its surface expression immunoglobulin molecules or the segmental any host cell of its antigen-specific.Proper host cell comprises the plasmoma clone of immunoglobulin (Ig) feminine gender.The example of this class clone includes, but are not limited to NS1 clone, Sp2/0 clone and P3 clone.Other suitable clones are conspicuous to those of ordinary skills.

(shown in Fig. 2 A and 2B) provides a kind of method in another preferred embodiment, wherein utilize transfection the host cell of construct come the indirect induction necrocytosis, the heterologous polynucleotide and the transcription regulatory region that comprise " suicide " gene in the described construct operably link together, and the latter is induced when the generation surface immumoglobulin is molecule crosslinked." suicide gene " is meant the nucleic acid molecule that causes necrocytosis when expressing.The polynucleotide that can be used as suicide gene comprise many necrocytosis induced sequences known in the art.Preferred suicide gene is a toxin-encoding, such as the ETA chain, and diphtheria toxin A chain, ricin A chain, abrin A chain, modeccin A chain and α sarcina (those genes of α-sarcin).A preferred suicide gene coding diphtheria toxin A subunit.In case the immunoglobulin molecules generation antigen induction of host cell surface is crosslinked, the promotor of apoptosis-inducing gene is just induced, thereby expresses suicide gene, promotes necrocytosis.

When adopting this method, can use can be in its surface expression immunoglobulin molecules or its antigen-specific fragment, and can identify any host cell of transcription regulatory region by express spectra, wherein said transcription regulatory region is induced when molecule crosslinked in that surface immumoglobulin takes place.Proper host cell comprises the plasmoma clone of early stage B cell lymphoma clone and immunoglobulin (Ig) feminine gender.The example of this class clone includes, but are not limited to CH33 clone, CH31 clone, WEHI-231 clone, NS1 clone, Sp2/0 clone and P3 clone.Other suitable clones are conspicuous to those of ordinary skills.

At host cell is in the situation of the negative plasmoma clone of Ig-, can make these cells by being attached on the solid substrate with the interaction that is combined in the plasmoma specific antibody on the matrix.Suitable plasmoma specific antibody comprises, but be not limited to anti-CD38 antibody (Yi, Q. etc., Blood90:1960-1967 (1997)), anti-CD31 antibody (Medina, F. etc., Cytometry39:231-234 (2000)), anti-CD20 antibodies (Haghighi, B. etc., Am.J.Hematol.59:302-308 (1998)) and anti-CD10 antibody (Dunphy, C.H., Acta.Cytol.40:358-362 (1996)).

Direct and the indirect antigen induction necrocytosis method of describing in the literary composition can be used in combination.For example, at host cell is in those embodiments of early stage B cell lymphoma and the direct cell death inducing of antigen cross-linking, can be by quickening the necrocytosis of antigen induction with the early stage B cell lymphoma host cell of a kind of like this construct transfection, the polynucleotide and the transcription regulatory region of the external cytotoxic T cell epi-position of coding operably link together in the described construct, and the latter is induced when the generation surface immumoglobulin is molecule crosslinked.When the antigen cross-linking cell contacts with specific cytotoxic T cells, necrocytosis will obtain accelerating.Similarly, at host cell is early stage B cell lymphoma, and antigen cross-linking directly influences in apoptotic those embodiments, can be by quickening the necrocytosis of antigen induction with the early stage B cell lymphoma host cell of a kind of like this construct transfection, suicide gene and transcription regulatory region operably link together in the described construct, and the latter is induced when the generation surface immumoglobulin is molecule crosslinked.

Heavy chain immunoglobulin can be modified, make specific antigen therein acceptor the signal of easy detection takes place to induce in the crosslinked cell through specific antigen.An embodiment preferred is to utilize the apoptosis-inducing system to select the cell killing that is caused because of the antigen expressed specific receptors.An example of apoptosis-inducing system comprises people FAS (CD95, APO-1) acceptor, it is a member of neoplasm necrosis-trk C superfamily, and this superfamily plays a role in regulating apoptosis by raising and assemble dead inducement signal complex body (this complex body activates a series of proteolysis caspases).There are several parts of reports to describe inducibility necrocytosis system based on FAS, wherein can come cell death inducing by chimeric protein, this chimeric protein contains the kytoplasm " death domain " of FAS, and is coupled at isoacceptor not, thereby can come cell death inducing by the various kinds of cell regulon.Ishiwatari-Hayasaka etc. successfully utilize the ectodomain of mouse CD44 and people FAS with the anti-CD44 antibody of multivalence cell death inducing (Ishiwatari-Hayasaka H. etc., J Immunol 163:1258-64 (1999)) when crosslinked taking place.In addition, cell death inducing (Takahashi T etc., J BiolChem271:17555-60 (1996)) when crosslinked can take place with anti-G-CSFR antibody in the chimeric human G-CSF R/FAS of proofs such as Takahashi (born of the same parents outer/kytoplasm) albumen.These authors are verified, and the chimeric protein dimer can not cell death inducing.Complex body must be a trimeric form at least.

In a preferred embodiment, made up a mosaic gene, wherein the carboxyl terminal of the CH1 structural domain of the membrane spaning domain of FAS and kytoplasm death domain and people IgM heavy chain merges (CH1-Fas, Figure 13 (a)).As described herein various VH genes are inserted this construct, thereby prepare VH-CH1-Fas recombinant chou vaccinia virus library.At the different light chain immunoglobulins or infected in the host cell of the recombinant chou vaccinia virus of the different light chain immunoglobulins of coding that psoralene was handled of encoding that infected VH-CH1-Fas construct and transfection, assembling has the membrane receptor of VH-CH1-Fas.The cell that those expression have required specific heavy chain and chain variable region gene combination will have the generation under the situation that autotelic specific immobilized antigen exists of some membrane receptor crosslinked.The formation of the functional complex body of VHCH1/FAS oligomer is with cell death inducing.Can be by crosslinked with polyvalent antigen or form tripolymer by the antigen more than 1 is fixed on tissue culturing plate or the pearl.

In the embodiment of an accommodation, in fusion rotein, express the VH library, comprise the membrane spaning domain of FAS and the polypeptide of kytoplasm death domain and the carboxyl terminal of IgM heavy chain CH4 structural domain in the described fusion rotein and merge (Figure 13 (b)).In another embodiment, the kytoplasm death domain of FAS is fused to the carboxyl terminal (Figure 13 (c)) of the membrane spaning domain of IgM heavy chain CH4 structural domain back.

Latter two embodiment (Figure 13 (b) and (c)) causes dimer synthon Fas death domain, and it can promote to form the needed trimer compositions of cell death inducing signal, thereby improves the quantity of selected antigen-specific receptor.But utilize monomer construct (Figure 13 (a)), can choose few but the antigen receptor that avidity is higher of quantity, reduce the background of non-antigen-specific cell death simultaneously.These the two kinds acceptor differences that have dimer Fas structural domain are that the film district of striding of encoding in the fusion rotein is Fas deutero-or IgM deutero-.The film district of striding derived from IgM may be more effective for the membrane receptor in the cell that is expressed in the bone-marrow-derived lymphocyte pedigree.An advantage of this embodiment is that it is not limited to the B cell.Specifically, monomeric Fas construct can comprise epidermic cell system, and Hela cell and BSC-1 cell etc. can produce and synthesize in the cell type widely of high titre vaccinia virus and be expressed as membrane receptor.

In another embodiment, provide a kind of screening method of the cell signaling based on antigen induction, be used for reclaiming coding immunoglobulin molecules or the segmental polynucleotide of its antigen-specific.According to this method, with reporter constructs (for example luciferase) transfection host cell that detects easily, this construct and transcription regulatory region operably link together, and the latter is raised when the generation surface immumoglobulin is crosslinked.To contact with antigen at its surface expression immunoglobulin (Ig) or its segmental host cell pond, take place when crosslinked, can be at this cell centralized detecting signal.With reference to first step of immunoglobulin (Ig) authentication method described above, can following enforcement signal transmission method.The first polynucleotide library of coding immunoglobulin (Ig) subunit polypeptide is divided into a plurality of ponds, for example about 2,5,10,25,15,75,100 or more a plurality of pond, each pond contains about 10,100,10 ³, 10 ⁴, 10 ⁵, 10 ⁶, 10 ⁷, 10 ⁸Or 10 ⁹Individual coding has the different polynucleotide of the immunoglobulin (Ig) subunit polypeptide of different variable regions.Preferred each pond begins to contain about 10 ³Individual polynucleotide.With each pond expansion, stay a replica to be used for the recovery of back.Be structured in virus vector (preferred poxvirus the polynucleotide group, more preferably vaccinia virus) in the situation, can use part wherein to prepare described pond by for example with the dilution of infectious titer library stoste with the micro-culture that hangs down MOI (for example MOI＜0.1) infected tissue's culturing cell.Usually through after 48 hours the infection, virus titer obtains the amplification more than 1000 times.Concentrate the amplicon virus titre to reduce separately because the danger that the growth relatively rapidly of more competitive subgroup causes the recombinant chou subgroup to lose a plurality of.

Infect the host cell pond that equates with prepared viral pond quantity with viral pond then.These host cells are expressed reporter molecule when crosslinked by through engineering approaches so that in that surface immumoglobulin takes place.The quantity of the host cell that infect in each pond depends on the quantity of these concentrated polynucleotide that contain and required MOI.The second polynucleotide library is also imported the host cell pond, and allow surface expression immunoglobulin molecules or its fragment at host cell.

Then the host cell pond is contacted under certain condition with purpose antigen, take place to express detectable reporter molecule when crosslinked at described immunoglobulin molecules at the host cell of its surface expression antigen specific immune globulin molecule under this condition, screen the reporter molecule expression in each host cell pond then.Gather in the crops those and can detect the host cell pond that reporter molecule is expressed, reclaim the polynucleotide in first library the sample that stays when beginning to increase this polynucleotide pond.

Polynucleotide for coding for antigens specific immunoglobulin subunit polypeptide in further enrichment first library are divided into a plurality of inferior ponds with the polynucleotide that are recovered to above.Setting up inferior pond makes it to contain still less different members than the pond of using above.For example, if each first pond contains 10 ³Individual different polynucleotide, each should on average contain about 10 or 100 different polynucleotide the inferior pond of foundation.The inferior pond and second library are imported host cell, allow film surface expression immunoglobulin molecules or its fragment at host cell.Above resembling then host cell is contacted with antigen, identify and to detect the inferior pond of those host cells that reporter molecule is expressed, the polynucleotide of concentrating recovery first library that duplicate as mentioned above from before having stayed.Those of ordinary skills understand, this process can be repeated again one or repeatedly so that the abundant polynucleotide of enrichment coding for antigens specific immunoglobulin subunit polypeptide.

In case finish the step of the polynucleotide in further selection and enrichment first library, and after separating these polynucleotide, carry out similar process and reclaim the polynucleotide in second library, they can the binding purposes specific antigen as an immunoglobulin molecules or the segmental part of its antigen-specific.

Any suitable reporter molecule may be used to this method, and used host cell is depended in its selection, can be for the detecting instrument that utilizes and the easy degree of detection.Suitable reporter molecule includes, but are not limited to luciferase, green fluorescent protein and beta-galactosidase enzymes.

Anyly can may be used to this method at the host cell of its surface expression immunoglobulin molecules.Preferred host cell comprises the plasmoma cell of immunoglobulin (Ig) feminine gender, NS1 cell for example, Sp2/0 cell or P3 cell, and early stage B cell lymphoma cell.

Similar with above-described necrocytosis method, kinetics is considered to require the expression of reporter constructs to occur in before the inducing of CPE.However, preferably owing to after occurring in host cell and antigen contacting about 1 hour to 4 days of the expression of crosslinked the detected reporter molecule that causes of the immunoglobulin molecules antigen induction on the host cell surface, so that take place prior to inducing of CPE.More preferably, after the expression of reporter molecule occurs in host cell and antigen contacts about 1 hour, 2 hours, about 3 hours, 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 28 hours, about 32 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours.More preferably the expression of reporter molecule occur in host cell contact with antigen the back about 12 hours in.

" derivative transcription regulatory region when surface immumoglobulin is molecule crosslinked " is meant a kind of like this zone, host cell promotor for example, and it regulates and control the gene that those immunoglobulin molecules at surface expression are raised when crosslinked usually.A preferred example of this class transcription regulatory region is the BAX promotor, and it is raised when B cell lymphoma cell generation surface immumoglobulin is molecule crosslinked in early days.

In another embodiment, provide a kind of selection coding immunoglobulin molecules or segmental selection of its antigen-specific or screening method, this method is based on the antigen-specific combination.Fig. 5 has shown this embodiment.According to this method, only depend on and detect antigen in conjunction with being recovered in its surface expression antigen specific immune globulin molecule or its segmental host cell.Antigen promptly utilizes the characteristics of conjugated antigen in conjunction with can be used as a kind of system of selection, by with the above-described host cell of directly selecting expression antigen specific immune globulin molecule based on the similar method of necrocytosis.For example, if antigen is combined on the solid substrate, can by be attached to by antigen reclaim on the solid substrate suspension can with antigen bonded host cell.Perhaps, antigen is in conjunction with can be used as screening process, promptly by with the above-mentioned similar method of cell signaling that is used for antigen induction the screening of antigen bonded being carried out detecting in the host cell pond.For example, will contact with antigen, detect given concentrated antigen in conjunction with situation, for example measure by detecting with antigen bonded enzyme-antibody coupling matter by immunoassay at surface expression immunoglobulin (Ig) or its segmental host cell pond.

With reference to first step of immunoglobulin (Ig) authentication method described above, the selection of can following enforcement carrying out via the antigen-specific combining method.Selection can be infected at the host cell of surperficial high level expression immunoglobulin molecules and/or transfection.Preferably, host cell suspension growth.After infecting as mentioned above with the first and second polynucleotide libraries, allow the synthetic and assembling of antibody molecule.Then host cell is transferred in the hole that is combined with antigenic microtiter plate on the surface.Thereby the host cell of energy conjugated antigen is attached to the surface in hole, removes unconjugated cell by soft washing.Perhaps, can reclaim the host cell of conjugated antigen by for example fluorescence-activated cell sorting (FACS).FACS is called the fluidic cell letter sorting again, can be used to select individual cells according to optical characteristics such as comprising fluorescence.It is useful screening the jumpbogroup cell in the short period of time.Reclaim those at last and combine antigenic host cell, thereby the polynucleotide of the coding first immunoglobulin (Ig) subunit polypeptide in enrichment first library, the described first immunoglobulin (Ig) subunit polypeptide can specific combination purpose antigen as an immunoglobulin molecules or the segmental part of its antigen-specific.

Polynucleotide in further selection and enrichment first library, and after separating these polynucleotide, process reclaims the polynucleotide in second library like the implementation of class, and they can be in conjunction with specific purpose antigen as an immunoglobulin molecules or the segmental part of its antigen-specific.

Anyly can may be used to this system of selection at the host cell of its surface expression immunoglobulin molecules.Preferred host cell comprises the plasmoma cell of immunoglobulin (Ig) feminine gender, NS1 cell for example, Sp2/0 cell or P3 cell, and early stage B cell lymphoma cell.Preferred these cells can suspension growth.

With reference to first step of immunoglobulin (Ig) authentication method described above, can followingly screen by the antigen-specific combining method.The first polynucleotide library that is structured in the coding immunoglobulin (Ig) subunit polypeptide in the virus vector is divided into a plurality of ponds by aforesaid method.Infect the host cell pond that equates with prepared viral pond quantity with viral pond then.In this screening method, preferred host cell adheres on the solid substrate.The second polynucleotide library is imported the host cell pond equally, allow in host's surface expressing immunoglobulin molecule or its fragment.

Then the host cell pond is contacted with purpose antigen.Behind the antigen incubation, the not conjugated antigen that flush away is excessive.The antigen that screens cell pool at last is in conjunction with situation.Can detect the antigen combination by several different methods.For example, antigen can be puted together a kind of enzyme.After removing unconjugated antigen, add substrate, and measurement of enzymatic reaction products.Utilize second antibody conjugate or Streptavidin/biotin system can strengthen this method.This class screening method is well known to those of ordinary skill in the art, and can obtain with kit form from regular businessman there.Equally, if antigen is combined on the displaing microparticle (for example gold bead), then can microscopic inspection antigen and the combining of host cell.The same with above-mentioned cell signaling method, gather in the crops those and detect and antigen generation bonded host cell pond, and reclaim the polynucleotide in first library that wherein comprises.Perhaps, identify and to detect antigen bonded host cell pond, reclaim the first library polynucleotide that wherein comprise the replica in this polynucleotide pond that stays when beginning to increase this polynucleotide pond.

The first library polynucleotide for further enrichment coding for antigens specific immunoglobulin subunit polypeptide are divided into a plurality of inferior ponds with the polynucleotide that are recovered to above.Setting up inferior pond makes it to contain still less different members than the pond of using above.For example, if each first pond contains 10 ³Individual different polynucleotide, each should on average contain about 10 or 100 different polynucleotide the inferior pond of foundation.Inferior pond is imported host cell with above-mentioned second library, make film surface expression immunoglobulin molecules or its fragment at host cell.Above resembling then host cell is contacted with antigen, results or identify simply and wherein can detect the inferior ponds of those host cells of antigen bonded, reclaim wherein comprise or replica in the first library polynucleotide that comprise.Those of ordinary skills understand, this process can be repeated again one or repeatedly so that the abundant polynucleotide of enrichment coding for antigens specific immunoglobulin subunit polypeptide.

further select and the polynucleotide in enrichment first library and separate these polynucleotide after, process reclaims the polynucleotide in second library like the implementation of class, they can be in conjunction with specific purpose antigen as an immunoglobulin molecules or the segmental part of its antigen-specific.

Anyly can may be used to this method at the host cell of its surface expression immunoglobulin molecules.Preferred host cell comprises the plasmoma cell of immunoglobulin (Ig) feminine gender, NS1 cell for example, Sp2/0 cell or P3 cell, and early stage B cell lymphoma cell.

When implementing the necrocytosis method of the direct and indirect antigen induction described in the literary composition, can purpose antigen be contacted with host cell by any method easily.For example, in certain embodiments, antigen (for example peptide or polypeptide) is attached on solid substrate.With in the text, " solid support " or " solid substrate " be known in the art can be in conjunction with cell or antigenic any type of upholder.The upholder of knowing comprises the plastics that are used for tissue culture, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and Mierocrystalline cellulose, polyacrylamide, gabbro and the magnetite modified.At goal of the invention, the character of carrier can be soluble to a certain extent or insoluble.In fact support material can have any possible node configuration, as long as the link coupled molecule can combine with cell.Therefore, the upholder configuration can be spherical, such as pearl; Or cylindrical, such as internal surface at test tube, the perhaps outside surface of bar.Perhaps, the surface can be flat such as sheet, test strip etc.Preferred upholder comprises the polystyrene pearl.Described upholder configuration comprises test tube, pearl, microballon, hole, plate, tissue culturing plate, flat board, microtest plate, microtiter plate, bottle, rod, band, bottle, agitator etc.Solid support can be a magnetic or nonmagnetic.Those skilled in the art will know that many other are applicable to, perhaps be easy to determine these materials in conjunction with cell or antigenic carrier.

Perhaps, can be at the described antigen of presenting cell surface expression of antigen expressed.With in the text, " presenting cell of antigen expressed " is meant at the antigenic cell of its surface expression purpose, and its phraseology makes this antigen to interact with the immunoglobulin molecules attached to host cell surface.The presenting cell of preferred antigen expressed is engineered the form of purpose antigen presentation for the reorganization body protein, but antigen also can be the natural antigen of this cell.Molecular biology and the protein expression technology that can utilize those of ordinary skills to know make up the reorganization presenting cell of described antigen expressed by any suitable method.Usually use the suitable cell of the coding antigenic plamid vector transfection of purpose, and the antigenic cell of desired polypeptides is expressed in screening.The antigenic reorganization presenting cell of preferred expression can stably be expressed purpose antigen.Presenting cell same type with antigen expressed is called " no antigen presenting cell " in the text but be not used to express the antigenic cell of purpose by through engineering approaches.Any suitable clone may be used to prepare the presenting cell of antigen expressed.The example of this class clone has included, but are not limited to transform the monkey kidney CVI system (COS-7, ATCC CRL1651) of SV40; Human embryo kidney (HEK) system (293, Graham etc., J.Gen Virol.36:59 (1977)); Baby hamster kidney cell (BHK, ATCC CCL10); Chinese hamster ovary cell DHFR (CHO, Urlaub and Chasin, Proc.Natl.Acad.Sci.USA77:4216 (1980)); Mouse podocyte (TM3, Mather, Biol.Reprod.23:243-251 (1980)); Monkey-kidney cells (CVIATCC CCL70); African green monkey kidney cell (VERO-76, ATCC CRL-1587); People's neck cancer cells (HELA, ATCC CCL2); Madin-Darby canine kidney(cell line) (MDCK, ATCC CCL34); Buffalo rat liver cell (BRL 3A, ATCC CRL1442); Human pneumonocyte (W138, ATCC CCL75); Human liver cell (hep G2, HB8065); Mouse breast cancer (MMT060562, ATCC CCL51); TRI cell (Mather etc., AnnalsN.Y.Acad.Sci383:44-68 (1982)); NIH/3T3 cell (ATCC CRL-1658) and mouse Lcell (ATCC CCL-1).Other clones are conspicuous to those of ordinary skills.(10801 UniversityBoulevard, Manassas VA20110-2209) can obtain various kinds of cell system from American type culture collection.

Those of ordinary skills understand that the presenting cell of antigen expressed also contains many natural antigenic determinants on its surface except purpose antigen.Estimate among the present invention that those express different widely immunoglobulin molecules from the teeth outwards or the segmental host cell of its antigen-specific can combine with these extra antigenic determinants.Therefore, use when having the antigenic antigen presentation presenting cell of purpose and contacting, be necessary at first to remove among the host cell group expression those host cells group of the activated immunoglobulin (Ig) of described other antigenic determinants with host cell of the present invention.The invention provides to be used for removing and to express the host cell that the self-faced antigen of no antigen presenting cell is had specific immunoglobulin molecules from the host cell group.Fig. 5 has shown this method.Taking it by and large, these methods are included in the host cell group with before the presenting cell of antigen expressed contacts, and the host cell group is contacted with no antigen presenting cell.

In one embodiment, this method comprises the host cell group is adsorbed onto on the no antigen presenting cell that is combined on the solid substrate.The polynucleotide that reclaim uncombined cell and/or wherein comprise contact host cell that is recovered to or the new host cell that has imported the polynucleotide that are recovered to then with the antigen presentation presenting cell.With in host cell pond and the system of selection that antigen contacts, the host cell pond just is adsorbed onto on the no antigen presenting cell that is combined on the solid substrate at those.Uncombined cell and/or the polynucleotide that wherein contain contacted host cell that is recovered to or the host cell that has imported the polynucleotide that are recovered to then during recovery obtained and closes with the antigen presentation presenting cell.

In another embodiment, described method comprises the host cell group is contacted under such condition with no antigen presenting cell, wherein express with no antigen presenting cell on those host cells of surface antigen reacted surface immunoglobulin molecules of antigenic determinant, immunoglobulin molecules on host cell surface takes place when crosslinked, the programmed cell death (for example apoptosis) that the front was described takes place, directly or indirectly necrocytosis or cell signaling (promptly expressing reporter molecule).Reclaim the host cell that those necrocytosis do not take place or do not express reporter molecule then, perhaps more particularly, retrieve polynucleotide from first or second library of those host cells.For example, if the host cell group of expressing immunoglobulin molecule is still attached on the solid substrate, and those cells that necrocytosis takes place discharge from matrix, and cell that maintenance adheres to and the polynucleotide that wherein comprised are reclaimed in then sucking-off and discard the content of nutrient solution.

Those of ordinary skills understand, remove the host cell of the immunoglobulin (Ig) of the antigenic determinant reaction that those are expressed and no antigen presenting cell carries among the host cell group, may need many wheels to eliminate.Our further imagination can with by the wheel enrichment like this some host cells replace eliminating by wheel, these cell expressings can with expressed purpose antigen-specific bonded immunoglobulin molecules on the antigen presentation presenting cell.

In another embodiment, provide a kind of screening method of the antigen-specific function based on immunoglobulin molecules to incorporate the polynucleotide of yard immunoglobulin molecules or its antigen-specific function fragment back and forth into own forces.According to this method, the host cell pond of completely soluble immunoglobulin molecules is expressed in preparation.Allow to express, detect the cell culture medium that obtains by the specific difference in functionality assay method of certain specific antigen of needs.According to this method, detected " function " can be the conventional effector function that immunoglobulin molecules is carried out, for example virus neutralization, and opsonization, ADCC, antagonist/agonist activity, histamine release, hemagglutination or hemagglutination suppress.Perhaps, described " function " only refers to conjugated antigen.

In a relevant embodiment, provide a kind of selection to have the known antigens specificity but the screening method of the immunoglobulin molecules that effector function changes.According to these embodiments, make up the immunoglobulin (Ig) subunit polypeptide library that has the known antigens specificity but in the constant domain of the given effector function of known participation, change.According to this method, the host cell pond of completely soluble immunoglobulin molecules is expressed in preparation.Allow to express, detect in the cell culture medium that obtains by the different functionalities assay method and improve or repressed activity.According to this method, detected " function " can be the performed conventional effector function of immunoglobulin molecules, for example virus neutralization, opsonization, complement combination, ADCC, antagonist/agonist activity, histamine release, hemagglutination or hemagglutination suppress.

With reference to first step of immunoglobulin (Ig) authentication method described above, screening that can following enforcement pairing effect subfunction.As mentioned above, with the coding complete excretory immunoglobulin (Ig) subunit polypeptide the first polynucleotide library be divided into a plurality of ponds, each pond contains about 10,100,10 ³, 10 ⁴, 10 ⁵, 10 ⁶, 10 ⁷, 10 ⁸Or 10 ⁹Individual coding has the different polynucleotide of the complete excretory immunoglobulin (Ig) subunit polypeptide of different variable regions.Preferred each pond begins to contain about 10 ³Individual polynucleotide.With each pond amplification, stay a replica to be used for the recovery of back.Be structured in virus vector (preferred poxvirus in the polynucleotide pond, more preferably vaccinia virus) in the situation, can use a part wherein to prepare described pond by for example with the dilution of infectious titer library stoste with the micro-culture that hangs down MOI (for example MOI＜0.1) infected tissue's culturing cell.Usually through after 48 hours the infection, virus titer obtains the amplification more than 1000 times.Concentrate the amplicon virus titre to reduce separately because the danger that the growth relatively rapidly of competitive subgroup causes the recombinant chou subgroup to lose a plurality of.

Infect the host cell pond that equates with prepared viral pond quantity with viral pond then.The quantity of the host cell that infect in each pond depends on the quantity of these concentrated polynucleotide that contain and required MOI.Basically, can use in the method can be by used viral vector infection, and can express any host cell of complete excretory immunoglobulin molecules.Preferred host cell comprises the plasmoma cell of immunoglobulin (Ig) feminine gender, NS1 cell for example, Sp2/0 cell, or P3 cell, and early stage B cell lymphoma cell.These cells can suspension culture or attached to solid surface.The second polynucleotide library is also imported the host cell pond, and allow to express complete excretory immunoglobulin molecules or its fragment.

Reclaim then and wherein cultivating the conditioned medium in host cell pond, detect by standardized function test and reply the antigenic effector function of special purpose.

Any suitable function test all can be used for this method.For example, can in virus neutralization test, detect in the cell conditioned medium liquid of gathering in the crops can in and the immunoglobulin molecules of target viral (for example HIV).Perhaps, can detect cell conditioned medium liquid blocking-up of gathering in the crops or the ability that promotes (promptly as antagonist or agonist) target cell function (for example apoptosis).Exemplary suitable function test has been described in the following examples.With in the text, " functional test " comprises that also the conventional ELISA assay method of for example utilizing those of ordinary skills to know detects the antigen combination simply.

When the conditioned medium in the given host cell pond of growing has required function, reclaim the polynucleotide in first library that comprises in the host cell in this pond the sample that stays when beginning to increase this polynucleotide pond.

Polynucleotide for coding for antigens specific immunoglobulin subunit polypeptide in further enrichment first library are divided into a plurality of inferior ponds with the polynucleotide that are recovered to above.Setting up inferior pond makes it to contain still less different members than the pond of using above.For example, if each first pond contains 10 ³Individual different polynucleotide, each should on average contain about 10 or 100 different polynucleotide the inferior pond of foundation.The inferior pond and second library are imported host cell as mentioned above, allow to express complete excretory immunoglobulin molecules or its fragment.Reclaim as mentioned above and wherein cultivating the conditioned medium in host cell pond, detect by standardized function test and to reply the antigenic effector function of special purpose, evaluation has the conditioned medium sample of required function characteristic, concentrate to reclaim the polynucleotide in first library that this pond, Asia host cell contained from duplicating of before having stayed.Those of ordinary skills understand, this process can be repeated again one or repeatedly so that the abundant polynucleotide of enrichment coding for antigens specific immunoglobulin subunit polypeptide.

In case finish the step of the polynucleotide in further selection and enrichment first library, and after separating these polynucleotide, carry out similar process and reclaim the polynucleotide in second library, they demonstrate required antigen-specific sexual function as immunoglobulin molecules or its segmental part.

Test kit.The present invention further provides the test kit that is used to select the antigen-specific recombinant chou immunoglobulin (Ig) of expression in the eukaryotic host cell.This test kit comprises one or more container, wherein fills to finish one or more required composition of method of describing in the literary composition.In one embodiment, this test kit comprises: (a) by operably connecting transcription regulatory region, the encode first polynucleotide library of a plurality of first immunoglobulin (Ig) subunit polypeptides, wherein each first immunoglobulin (Ig) subunit polypeptide comprises: first constant region for immunoglobulin that (i) is selected from CH and constant region of light chain, (ii) with the corresponding immune globulin variable region of described first constant region, and (iii) can instruct on the cell surface of the described first immunoglobulin (Ig) subunit polypeptide and express or excretory signal skin, wherein said first library construction is in the eucaryon virus vector; (b) by operably connecting transcription regulatory region, the encode second polynucleotide library of a plurality of second immunoglobulin (Ig) subunit polypeptides, wherein each second immunoglobulin (Ig) subunit polypeptide comprises: second constant region for immunoglobulin that (i) is selected from CH and constant region of light chain, wherein this second constant region for immunoglobulin is different with first constant region for immunoglobulin, (ii) with the corresponding immune globulin variable region of described second constant region, and (iii) can instruct the described second immunoglobulin (Ig) subunit polypeptide on cell surface, to express or the excretory signal peptide, the many skins of the wherein said second immunoglobulin (Ig) subunit can make up with the first immunoglobulin (Ig) subunit polypeptide, thereby form the surface immumoglobulin molecule, or its antigen-specific fragment, be attached on the host cell membrane, and this second library construction is in the eucaryon virus vector; (c) can express the host cell group of described immunoglobulin molecules.In this test kit, described first and second libraries all are that the form with the virion of infectious viral particle and deactivation provides, the virion energy host cells infected of wherein said deactivation makes its express the polypeptide that is wherein comprised, but does not carry out virus replication.In addition, can express can be by the antigen specific immune globulin molecule of selecting with antigenic interaction for the host cell that provides in the test kit.The method of describing in the purposes of this test kit and the literary composition conforms to.In certain embodiments, test kit comprises that contrast antigen and reagent are so that make the antigenic choice criteriaization of specific purpose.

Isolating immunoglobulin (Ig).Isolating antigen specific immune sphaeroprotein or its fragment have been the present invention further provides by any method preparation of describing in the literary composition.These isolating immunoglobulin (Ig)s can be as diagnosis or treatment reagent.The invention provides the composition that comprises isolating immunoglobulin (Ig) of the present invention and pharmaceutical acceptable carrier in addition.

Unless otherwise noted, implement the present invention and will adopt cytobiology, cell cultures, molecular biology, genetically modified organism is learned, microbiology, recombinant DNA and immunologic routine techniques, these all are the ordinary skills of this area.These technology have sufficient explanation in the literature.Referring to for example, Molecular Cloning A Laboratory Mannual, 2 ^NdEd., Sambrook etc., Cold Spring Harbor Laboratory Press (1989); Molecular Cloning:A Laboratory Mannual, Sambrook etc., ColdSpring Harbor Laboratory, New York (1992), DNA Cloning, volume I and II (D.N.Glover ed., 1985); Oligonucleotide Synthesis (M.J.Gait ed.1984); Mullis etc., United States Patent (USP) 4683195; Nucleic AcidHybridization (B.D.Hames﹠amp; S.J.Higgins ed.1984); Transcription and Translation (B.D.Hames﹠amp; S.J.Higginsed.1984); Culture of Animal Cells (R.I.Freshney, Alan R.Liss, Inc.1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); Thetreatise, and Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J.H.Miller and M.P.Calos eds.1987, Cold Spring Harbor Laboratory); MethodsIn Enzymology, Vol.154 and 155 (editor such as Wu), ImmunochemicalMethods In Cell And Molecular Biology (Mayer and Walker edit, Academic Press, London, 1987); Handbook of ExperimentalImmunology, VolI-IV (D.M.Weir and C.C.Blackwell edit, 1986); Manipulating the Mouse Embryo (Coid Spring Harbor LabortoryPress, Coid Spring Harbor, N.Y.1986); With Ausubel etc., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).

Antibody Engineering, 2 ^NdEdition, C.A.K.Borrebaeck, ed. has described the General Principle of antibody engineering among the Oxford Univ.Press (1995).ProteinEngineering, A Practical Approach, Richwood, D. etc., Eds., IRLPress at Oxford Univ.Press, Oxford has proposed the General Principle of protein engineering among the Eng. (1995).Nisonoff, A., Molecular Immunology, 2 ^NdEd, Sinauer Associates, Sunderland, MA (1984); And Steward, M.W., Antibodies, Their Structure and Function, Chapman and Hall, New York has proposed antibody and incomplete antibody bonded ultimate principle respectively among the NY (1984).In addition, the immunology ordinary method of specifically not describing known in the art is usually with reference to CurrentProtocols in Imunology, John Wiley﹠amp; Sons, New York; Stites etc. (editor), Basic and Clinical-Immunology (8 ^ThEd), Appleton﹠amp; Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds), SelectedMethods in Cellular Immunology, W.H.Freeman and Co., NewYork (1980) carries out.

The canonical reference document that proposes the immunology ultimate principle comprises Current Protocols inImmunology, John Wiley﹠amp; Sons, New York; Klein, J., Immunology:The Science of Self-Nonself Discrimination, John Wiley﹠amp; Sons, New York (1982); Kennett, R. etc., Monoclonal Antibodies, Hybridoma:A New Dimension in Biological Analyses, PlenumPress, New York (1980); Campbell, A., " Monoclonal AntibodyTechnology " in Burden, editors such as R., Laboratory Techniques inBiochemistry and Molecular Biology, Vol13, Elsevere, Amsterdam (1984).

Embodiment

Embodiment 1 makes up the diversified human normal immunoglobulin of specificity library

The polynucleotide library of following generation coding panimmunity sphaeroprotein subunit polypeptide.Gene by pcr amplification coding VH (variable region of heavy chain), VK (K variable region of light chain) and VL (going into variable region of light chain).For each of three kinds of variable region gene families, construction recombination plasmid library and vaccinia virus library.In the transfer/expression plasmid of variable region gene insertion, be between the immunoglobulin (Ig) leader sequence and constant region sequence of corresponding heavy chain or light chain based on p7.5/tk.Use this plasmid, generate corresponding vaccinia virus reorganization part, also can enter and behind the cell of vaccinia virus infection, directly utilize this plasmid high level expression immunoglobulin chain in transfection by three molecular recombination.At first infect lymphoma cell, use plasmid light chain library transient transfection then with bovine vaccine heavy chain library.The coexpression of IgM and light chain will cause the assembling and the surface expression of antibody molecule.

1.1 press pVHE.Make up the expression vector that comprises people μ membrane immunoglobulin constant region as described below, be named as PVHE in this article.This strategy as shown in Figure 3.Utilization can be from Clontech, Palo Alto, the SMART that CA obtains ^TMRACE cDNA amplification kit is from the cDNA of marrow RNA separation coding film mating type people IgM heavy chain.Utilize following quoting to carry out PCR:5 ' primer (huC μ 5B)/5 '-ATTAGGATCCGGTCACCGTCTCCTCAGGG-3 ' (SEQ ID NO:24) and 3 ' primer (huC μ 3S), 5 '-ATTAGTCGACTCATTTCACCTTGAACAAGGTGAC-3 ' (SEQ ID NO:25).Then the PCR product is inserted BamHI and the SalI site of pBluescript II/KS,, eliminate 2 BstEII sites between CH2 and CH4 structural domain so that carry out site-directed mutagenesis.Selecting not change the coded amino acid whose Nucleotide of these site replaces.

By following method, will be as Zauderer, PCT publication number WO 00/028016 and the plasmid p7.5/tk that hereinafter generates described in the embodiment 5 change into pVHE.Multiple clone site (MCS) with among the box replacement P7.5/tk that comprises following restriction site: NotI-NcoI-BssHII-BstEII-SalI generates p7.5/tk2.Described box with sequence 5 '-GCGGCCGCAAACCATGGAAAGCGCGCATATGGTCACCAAAAGTCGAC-3 ' is known as SEQ ID NO:26 in this article.Coding is cloned between NcoI and BssHII site in the p7.5/tk2 corresponding to the box of the signal peptide sequence of IgM heavy chain the-19--3 amino acids, generates p7.5/tk2L.Then will as above-mentioned generation, through the IgM of BstEII mutagenesis heavy chain clone p7.51tk ₂Among the BstEII among the L and the SalI site between, generate pVHE.Then, variable region of heavy chain (VH) box that generate through PCR, that comprise the Nucleotide of coded amino acid-4 to 110 as described below is cloned between the BssHII and BstEII site of pVHE, generates the polynucleotide library of coding film mating type heavy chain.Because overlapping between μ sequence of heavy chain and the selected restriction enzyme sites, this will cause that the film mating type heavy chain immunoglobulin subunit polypeptide that adjoins expresses with correct translation reading frame.

1.2 be called the expression vector that comprises people μ secretory immunoglobulin constant region of pVHE in the following structure literary composition of pVHE..Fig. 8 has shown should strategy.Utilize SMART RACE cDNAAmplification Kit from marrow RNA, to separate the cDNA of coding secretor type people IgM heavy chain.Upstream primer huC μ 5B holds BamHI site and the BstEII site of containing interpolation 5 ', is thereafter the amino acid/11 11-113 of VH and first amino acid of C μ H1.Downstream primer shuC μ 3S contains last 6 amino acid of secretor type C μ, is a terminator codon and SalI site subsequently.These primers have following sequence:

HuC μ 5B:5 '-ATTAGGATCCGGTCACCGTCTCCTCAGGG-3 ' (SEQ IDNO:27); With

shuCμ3S：5’-ATTAGTCGACTCAGTAGCAGGTGCCAGCTGT-3’(SEQ IDNO：28).

Rite-directed mutagenesis is carried out in the BamHI and the SalI site of the PCR product being inserted pBluescriptII/KS then, so that remove two BstEII sites that are positioned at CH2 and CH4 structural domain.The Nucleotide of selecting these site amino acids coding not change replaces.

Plasmid p7.5/tk2L with the preparation of 1.1 parts is converted into pVHEs by the following method.The secretor type IgM heavy chain of the BstEII sudden change that will prepare as mentioned above then is cloned into BstEII and the SalI site among the p7.5/tk2L, thereby produces pVHEs.The polynucleotide library that produces coding secretor type heavy chain will be cloned between the BssHII and BstEII site among the pVHEs then by variable region of heavy chain (VH) box that comprises the Nucleotide of coded amino acid-4 to 110 of PCR described below preparation.Because overlapping between μ sequence of heavy chain and the selected restriction enzyme sites causes expressing the secretor type heavy chain immunoglobulin subunit polypeptide that adjoins with correct translation frame.

1.3 the expression vector that comprises people κ and lambda immunoglobulin constant region of light chain of called after pVKE and pVLE in the following structure literary composition of pVKE and pVLE..Fig. 4 has shown this strategy.

(a) by the following method plasmid p7.5/tk is converted into pVKE.Connect two XhoI and two HindIII sites of removing p7.5/tk by filling and leading up, (one in main chain with 3 ApaLI sites through standard method, one is positioned at ColEl ori, another is at Amp) remove, substitute the multiple clone site (MCS) of p7.5/tk: NotI-NcoI-ApaLI-XhoI-HindIII-SalI with the box that contains following restriction site, thereby produce p7.5/tk3.This box is called SEQ ID NO:29 in the text, and its sequence is 5 '-GCGGCCGCCC ATGGATACGTGCACTTGACT CGAGAAGCTT AGTAGTCGAC-3 '.

Amino acid-19 in the coding κ light chain is cloned among the p7.5/tk3 between the NcoI and ApaLI site to the box of-2 pairing signal peptide sequences, produces p7.5/tk3L.Utilize SMART ^TMRACE cDNA Amplification Kit separates the cDNA in coding C κ district as mentioned above from marrow RNA, the primer is included in 5 ' the terminal XhoI site in the zone of coded amino acid 104-107+Ck, the SalI of terminator codon and 3 ' end.These primers have following sequence: huC κ 5:5 '-CAGGACTCGA GATCAAACGA ACTGTGGCTG-3 ' (SEQ ID NO:30); HuC κ 3:5 ' AATATGTCGA CCTAACACTCTCCCCTGTTG AAGCTCTTT-3 ' (SEQ ID NO:31); And huCk3:5 '-AATATGTCGA CCTAACACTC TCCCCTGTTG AAGCTCTT-3 ' (SEQ ID NO:32).Then C κ box is cloned between the XhoI and SalI site of p7.5/tk3L, produces pVKE.To be cloned among the pVKE between the ApaLI and XhoI site by kappa light chain variable district box (VK) that comprises the Nucleotide of coded amino acid-3 to 105 of PCR as described below preparation then.Because it is overlapping that κ sequence of light chain and selected restriction enzyme sites exist, cause expressing the κ light chain immunoglobulin (Ig) subunit polypeptide that adjoins with the correct translation frame.

(b) by the following method plasmid p7.5/tk3L is converted into pVLE.Utilize SMART ^TMRACE cDNA Amplification Kit separates the cDNA in coding C κ district as mentioned above from marrow RNA, the primer has the HindHIII site and the zone of the amino acid/11 05 to 107 of the V λ that encodes at 5 ' end, comprises terminator codon and SalI site at 3 ' end.These primers have following sequence: huC λ 5:5 '-ATTTAAGCTT ACCGTCCTACGAACTGTGGC TGCACCATCT-3 ' (SEQ ID NO:33); With huC λ 3 (SEQ IDNO:31). then C κ box is inserted HindIII and SalI site among the p7.5/tk3L, produce pVLE.To be cloned among the pVLE between the ApaLI and HindIII site by lambda light chain variable district box (VL) that comprises the Nucleotide of coded amino acid-3 to 140 of PCR as described below preparation then.Because it is overlapping that lambda light chain sequence and selected restriction enzyme sites exist, cause adjoining lambda light chain immunoglobulin (Ig) subunit polypeptide with the expression of correct translation frame.

1.4 variable region.By the following method, separate heavy chain through PCR, κ light chain and lambda light chain variable district are used for being cloned into the expression vector of preparation as mentioned above.Separation since a plurality of donor harvestings to normal people's marrow RNA (Clontech is on sale) be used for synthetic cDNA.The aliquot sample of cDNA prepared product is used for pcr amplification, and primer is to being selected from following a few cover primer: VH/JH, VK/JK or VL/JL.Table 1 and 2 has been listed the primer of the variable region that is used to increase.

(a) variable region of heavy chain.Because the design of plasmid expression vector, VH primer (forward primer of the primer centering in the heavy chain V district that promptly is used to increase) has following general configuration, and wherein the BssHII restriction site shows with underscore:

The VH primer: GCGCGCThe beginning of ACTCC-VH FR1 primer.

Design last 4 the amino acid whose codons of leader sequence of encoding that comprise of primer, wherein BssHII site coded amino acid-4 is followed by the special FR1 sequence of VH family to-3.Table 1 and 2 has been listed the sequence of different family specificity VH primers.Because last 5 amino acid of variable region of heavy chain (are amino acid/11 09-113, in 6 kinds of people's heavy chain J districts is identical) be included among the plasmid pVHE, the JH primer reverse primer of variable region of heavy chain (promptly be used to increase) thus have the BstEII site (underscore demonstration) that following configuration comprises coded amino acid 109 to 110:

The coding nucleotide sequence of the amino acid/11 03-108 of JH primer :-VH (finishing) with G- GTCACC

Utilize these primers, VH PCR product begins with the codon of coded amino acid-4 to 110, and wherein BssHII is an amino acid-4 and-3, finishes with the BstEII site of the codon that is positioned at amino acid/11 09 and 110.After suitable restriction enzyme digestion, these PCR product cloning are arrived in the pVHE that digested with BssHII and BstEII.

For most possible rearrangement variable region of heavy chain that increase, use VH and the JH primer family shown in the table 1 and 2.VH1,3 and 4 families account for 44 kinds in 51 kinds of V districts that exist in the people's gene group.The codon that comprises coded amino acid 109-113 in the expression vector has just been got rid of the single general JH primer of use.But, 5 the JH primers of each VH primer shown in can combined statement 1 and 2 are reduced the number of times of required PCR reaction.

(b) kappa light chain variable district.The VK primer, the forward primer in the kappa light chain variable district that promptly is used to increase has following general configuration, and wherein underscore has shown the ApaLI restriction site:

The VK primer: GTGCACThe beginning of TCC-V κ FR1 primer

The VK primer contains last 3 the amino acid whose codons of coding κ light chain leader sequence, and wherein ApaLI site coded amino acid-3 and-2 is followed by VK family specificity FR1 sequence.Because the codon of coding kappa light chain variable last 4 amino acid in district (amino acid/11 04-107) is included among the expression vector pVKE, so the JK primer, the reverse primer in the kappa light chain variable district that promptly is used to increase represents following configuration:

The JK primer:

The nucleotide sequence of the amino acid 98-103 of-coding VK- CTCGAG

XhoI site (showing with underscore) comprises the codon of the amino acid/11 04-105 in coding kappa light chain variable district.The PCR product in coding kappa light chain variable district begins with the codon of amino acid-3, finishes with the codon of amino acid/11 05, wherein has the ApaLI site of the codon that comprises amino acid-3 and-2 and comprises the XhoI site of the codon of amino acid/11 04 and 105.VK1/4 and VK3/6 primer respectively have two degeneracy nucleotide sites.Utilize these JK primers (referring to table 1 and 2), the sudden change of Val to Leu will take place at amino acid/11 04 place in JK1,3 and 4, and the sudden change of Asp to Glu takes place at amino acid/11 05 place JK3.

(c) lambda light chain variable district.The VL primer, the forward primer of the primer centering in the lambda light chain variable district that promptly is used to increase has following general configuration, and wherein the ApaLI restriction site shows with underscore:

The VL primer: GTGCACThe beginning of TCC-VL

Described ApaLI site comprises the codon of coded amino acid-3 and-2, follows by VL family specificity FR1 sequence.Because the codon of coding last 5 amino acid of VL (amino acid/11 03-107) is included among the expression vector pVLE,, thereby comprise the HindIII site (with underscore demonstration) of the codon that contains coded amino acid 103-104 so the JL primer demonstrates following configuration:

The JL primer: the nucleotide sequence of amino acid 97-102 among the-VL- AAGCTT

The PCR product in coding lambda light chain variable district begins with the codon of amino acid-3, and with the codon end of amino acid/11 04, ApaLI site wherein comprises the codon of amino acid-3 and-2, and the HindIII site comprises the codon of amino acid/11 03 and 104.

Table 1. is used for the Oligonucleolide primers of pcr amplification human normal immunoglobulin variable region.The recognition site of the restriction enzyme that the clone uses shows with boldface letter.Primer sequence from 5 ' to 3 '.

VH1(SEQ ID NO：34)	TTT TGC GCG CAC TCC CAG GTG CAG CTG GTG CAG TCT GG
		VH2(SEQ ID NO：144)	AATA TGC GCG CAC TCC CAG GTC ACC TTG AAG GAGTCT GG
VH3(SEQ ID NO：35)	TTT TGC GCG CAC TCC GAG GTG CAG CTG GTG GAG TCT GG
		VH4(SEQ ID NO：36)	TTT TGC GCG CAC TCC CAG GTG CAG CTG CAG GAG TCG GG
VH5(SEQ ID NO：145)	AATA TGC GCG CAC TCC GAG GTG CAG CTG GTG CAG TCT G

JH1(SEQ ID NO：37)	GAC GGT GAC CAG GGT GCC CTG GCC CCA
		JH2(SEQ ID NO：38)	GAC GGT GAC CAG GGT GCC ACG GCC CCA
JH3(SEQ ID NO：39)	GAC GGT GAC CAT TGT CCC TTG GCC CCA
		JH4/5(SEQ ID NO：40)	GAC GGT GAC CAG GGT TCC CTG GCC CCA
JH6(SEQ ID NO：41)	GAC GGT GAC CGT GGT CCC TTG GCC CCA
VK1(SEQ ID NO：42)	TTT GTG CAC TCC GAC ATC CAG ATG ACC CAG TCT CC
		VK2(SEQ ID NO：43)	TTT GTG CAC TCC GAT GTT GTG ATG ACT CAG TCT CC
VK3(SEQ ID NO：44)	TTT GTG CAC TCC GAA ATT GTG TTG ACG CAG TCT CC
		VK4(SEQ ID NO：45)	TTT GTG CAC TCC GAC ATC GTG ATG ACC CAG TCT CC
VK5(SEQ ID NO：46)	TTT GTG CAC TCC GAA ACG ACA CTC ACG CAG TCT CC
		VK6(SEQ ID NO：47)	TTT GTG CAC TCC GAA ATT GTG CTG ACT CAG TCT CC
		JK1(SEQ ID NO：48)	GAT CTC GAG CTT GGT CCC TTG GCC GAA
JK2(SEQ ID NO：49)	GAT CTC GAG CTT GGT CCC CTG GCC AAA
		JK3(SEQ ID NO：50)	GAT CTC GAG TTT GGT CCC AGG GCC GAA

JK4(SEQ ID NO：51)	GAT CTC GAG CTT GGT CCC TCC GCC GAA
		JK5(SEQ ID NO：52)	AAT CTC GAG TCG TGT CCC TTG GCC GAA
		VL1(SEQ ID NO：53)	TTT GTG CAC TCC CAG TCT GTG TTG ACG CAG CCG CC
VL2(SEQ ID NO：54)	TTT GTG CAC TCC CAG TCT GCC CTG ACT CAG CCT GC
		VL3A(SEQ ID NO：55)	TTT GTG CAC TCC TCC TAT GTG CTG ACT CAG CCA CC
VL3B(SEQ ID NO：56)	TTT GTG CAC TCC TCT TCT GAG CTG ACT CAG GAC CC
		VLA(SEQ ID NO：57)	TTT GTG CAC TCC CAC GTT ATA CTG ACT CAA CCG CC
VL5(SEQ ID NO：58)	TTT GTG CAC TCC CAG GCT GTG CTC ACT CAG CCG TC
		VL6(SEQ ID NO：59)	TTT GTG CAC TCC AAT TTT ATG CTG ACT CAG CCC CA
VL7(SEQ ID NO：60)	TTT GTG CAC TCC CAG GCT GTG GTG ACT CAG GAG CC
JL1(SEQ ID NO：61)	GGT AAG CTT GGT CCC AGT TCC GAA GAC
		JL2/3(SEQ ID NO：62)	GGT AAG CTT GGT CCC TCC GCC GAA T

Table 2. is used for the Oligonucleolide primers of pcr amplification human normal immunoglobulin variable region.The recognition site of the restriction enzyme that the clone uses shows with boldface letter.Primer sequence is with from 5 ' to 3 ' direction indication.

VH1a(SEQ ID NO：63)	AATA TGC GCG CAC TCC CAG GTG CAG CTG GTG CAG TCT GG
		VH2a(SEQ ID NO：64)	AATA TGC GCG CAC TCC CAG GTC ACC TTG AAG GAG TCT GG
VH3a(SEQ ID NO：65)	AATA TGC GCG CAC TCC GAG GTG CAG CTG GTG GAG TCT GG
		VH4a(SEQ ID NO：66)	AATA TGC GCG CAC TCC CAG GTG CAG CTG CAG GAG TCG GG
VH5a(SEQ ID NO：67)	AATA TGC GCG CAC TCC GAG GTG CAG CTG GTG CAG TCT G
JH1a(SEQ ID NO：68)	GA GAC GGT GAC CAG GGT GCC CTG GCC CCA
		JH2a(SEQ ID NO：69)	GA GAC GGT GAC CAG GGT GCC ACG GCC CCA
JH3a(SEQ ID NO：70)	GA GAC GGT GAC CAT TGT CCC TTG GCC CCA
		JH4/5a(SEQ ID NO：71)	GA GAC GGT GAC CAG GGT TCC CTG GCC CCA
JH6a(SEQ ID NO：72)	GA GAC GGT GAC CGT GGT CCC TTG GCC CCA
VK1a(SEQ ID NO：73)	CAGGA GTG CAC TCC GAC ATC CAG ATG ACC CAG TCT CC
		VK2a(SEQ ID NO：74)	CAGGA GTG CAC TCC GAT GTT GTG ATG ACT CAG TCT CC

VK3a(SEQ ID NO：75)	CAGGA GTG CAC TCC GAA ATT GTG TTG ACG CAG TCT CC
		VK4a(SEQ ID NO：76)	CAGGA GTG CAC TCC GAC ATC GTG ATG ACC CAG TCT CC
VK5a(SEQ ID NO：77)	CAGGA GTG CAC TCC GAA ACG ACA CTC ACG CAG TCT CC
		VK6a(SEQ ID NO：78)	CAGGA GTG CAC TCC GAA ATT GTG CTG ACT CAG TCT CC
		JK1a(SEQ ID NO：79)	TT GAT CTC GAG CTT GGT CCC TTG GCC GAA
JK2a(SEQ ID NO：80)	TT GAT CTC GAG CTT GGT CCC CTG GCC AAA
		JK3a(SEQ ID NO：81)	TT GAT CTC GAG TTT GGT CCC AGG GCC GAA
JK4a(SEQ ID NO ：82)	TT GAT CTC GAG CTT GGT CCC TCC GCC GAA
		JK5a(SEQ ID NO：83)	TT AAT CTC GAG TCG TGT CCC TTG GCC GAA
		VL1a(SEQ ID NO：84)	CAGAT GTG CAC TCC CAG TCT GTG TTG ACG CAG CCG CC
VL2a(SEQ ID NO：85)	CAGAT GTG CAC TCC CAG TCT GCC CTG ACT CAG CCT GC
		VL3Aa(SEQ ID NO：86)	CAGAT GTG CAC TCC TCC TAT GTG CTG ACT CAG CCA CC
VL3Ba(SEQ ID NO：87)	CAGAT GTG CAC TCC TCT TCT GAG CTG ACT CAG GAC CC
		VL4a(SEQ ID NO：88)	CAGAT GTG CAC TCC CAC GTT ATA CTG ACT CAA CCG CC

VL5a(SEQ ID NO：89)	CAGAT GTG CAC TCC CAG GCT GTG CTC ACT CAG CCG TC
		VL6a(SEQ ID NO：90)	CAGAT GTG CAC TCC AAT TTT ATG CTG ACT CAG CCC CA
VL7a(SEQ ID NO：91)	CAGAT GTG CAC TCC CAG GCT GTG GTG ACT CAG GAG CC
JL1a(SEQ ID NO：92)	AC GGT AAG CTT GGT CCC AGT TCC GAA GAC
		JL2/3a(SEQ ID NO：93)	AC GGT AAG CTT GGT CCC TCC GCC GAA TAC

Embodiment 2

Selection strategy in conjunction with the human normal immunoglobulin of specific antigen

Following selection comprises the vaccinia virus expression vector of the polynucleotide of coding recombinant chou heavy chain immunoglobulin subunit polypeptide, can give specificity to specific antigen when described immunoglobulin (Ig) subunit polypeptide and some unidentified light chain make up, as shown in Figure 1.The selection of specific immune sphaeroprotein heavy chain and light chain divides two stages to finish.At first, utilize pVHE to be transferring plasmid,, in based on the carrier of poxvirus, make up from the diversified heavy chain library of the antibody produced cell of immunity or immune donor by three molecular recombination (seeing embodiment 5) as structure as described in the embodiment 1; Structure as described in embodiment 1 such as plasmid vectors such as pVKE and pVLE in make up the similar library of light chain immunoglobulin, wherein the expression of recombinant gene is by p7.5 bovine vaccine promoter regulation.Immunoglobulin heavy chain constant region in the poxvirus construct is designed to keep membrane spaning domain, causes expressing on surface film immunoglobulin receptor.With infection multiplicity is the poxvirus heavy chain library host cells infected of 1 (MOI=1), for example early stage B cell lymphoma cell.After 2 hours, allowing each cell can absorb and express under the condition of average 10 or how independent light chain plasmid transfection infected cells with the light chain plasmid library.Because the expression of recombinant gene is subjected to the regulation and control of vaccinia virus promotor in this plasmid, so recombinant gene product high level expression in the kytoplasm of the cell that has infected vaccinia virus is integrated and need not nuclear.Under these conditions, individual cells can be expressed multiple antibody, and wherein different light chains are combined with identical heavy chain, forms distinctive H2L2 structure in each infected cell.

2.1 direct antigen induction sexual cell apoptosis.With the early stage B cell lymphoma host cell of recombinant chou vaccinia virus infection of coding recombinant chou heavy chain immunoglobulin subunit polypeptide, and carry out transfection with the plasmid of described coding recombinant chou light chain immunoglobulin (Ig) subunit polypeptide.Host cell is replied the crosslinked generation of antigen specific immunoglobulin acceptor by inducing the death of spontaneous growth-inhibiting and programmed cellization.General introduction as Fig. 1; allow the synthetic and assembling of antibody molecule to carry out 12 hours or longer; specific antigens is presented on synthetic particle or polymkeric substance in this process; perhaps on the surface of antigen presentation cell; so that take place crosslinkedly with any specific immunoglobulin acceptor, and induce the apoptosis of selected antigen presentation indicator cells.Extraction enrichment coding in the recombinant chou vaccinia virus gene group of being induced the apoptotic cell of generation is given the polynucleotide of required specific immunoglobulin heavy chain gene.

2.2 indirect antigen induction cell death.Shown in Fig. 2 A (following hurdle) and 2B (going up the hurdle), with the early stage B cell lymphoma host cell of construct transfection, the promotor of apoptosis-inducing gene in this construct (BAX promotor) drives the expression of external cytotoxic T cell epi-position.Host cell is expressed the CTL epi-position when replying antigen specific immune sphaeroprotein acceptor crosslinked, these crosslinked cells when adding special CTL the hydrolysis phenomenon will take place.The plasmid of the coding of the host cell of these stable transfections recombinant chou vaccinia virus infection of recombinant chou heavy chain immunoglobulin subunit polypeptide that is encoded, and transfection then recombinant chou light chain immunoglobulin (Ig) subunit polypeptide.Generalized as Fig. 1 institute; allow the synthetic and assembling of antibody molecule to carry out 12 hours or longer; specific antigens is presented on synthetic particle or polymkeric substance in this process, perhaps on the surface of antigen presentation cell, so that it is crosslinked that any specific immunoglobulin acceptor is taken place.When adding epitope specificity CTL, crosslinked cell generation hydrolysis phenomenon takes place in those surface immumoglobulin molecules, thus the indirect induction necrocytosis.

2.3 direct antigen induction cell death.Shown in Fig. 2 A (going up the hurdle) and 2B (following hurdle), with the early stage B cell lymphoma host cell of construct transfection, the promotor of apoptosis-inducing gene in this construct (BAX promotor) is driving the expression of the cytotoxicity A subunit of diphtheria toxin.Host cell is expressed the toxin subunit when replying antigen specific immune sphaeroprotein acceptor crosslinked, these crosslinked cells will experience necrocytosis.The plasmid of the coding of the host cell of these stable transfections recombinant chou vaccinia virus infection of recombinant chou heavy chain immunoglobulin subunit polypeptide that is encoded, and transfection then recombinant chou light chain immunoglobulin (Ig) subunit polypeptide.Generalized as Fig. 1 institute; synthetic and the assembling of antibody molecule was carried out 12 hours or longer; specific antigens is presented on synthetic particle or polymkeric substance in this process, perhaps on the surface of antigen presentation cell, so that it is crosslinked that any specific immunoglobulin acceptor is taken place.Crosslinked cell takes place and experiences necrocytosis rapidly and directly in those surface immumoglobulin molecules.

2.4 discuss.The reason that the surperficial Ig acceptor that the expression of these recombinant genes is crosslinked raises is, each the expression of two constructs is all by a kind of like this promoter regulation of gene, and this gene crosslinked back of Ig that is expressed in the B cell lymphoma cell is in early days raised naturally.This can illustrate by the BAX promotor.BAX is the example of proapoptosis gene (proapoptotic gene), and they are raised in the B cell lymphoma cell usually under these conditions in early days.The control region of other genes (promotor) may show equally good or better.Can identify this genoid by for example more early stage B cell lymphoma cell gene expression profile before and after the cross linking membrane Ig on microarray.

Use the construct transfectional cell, make it to express diphtheria A chain (dipA), than only passing through the crosslinked generation of Ig apoptosis more rapidly.Can induce necrocytosis more rapidly by the specific cytotoxic T cells that adds some target peptide, the natural MHC molecule of expressing on described target peptide and this cell is relevant, is the minigene coding that is subjected to BAX or class BAX promoter regulation by its expression.In addition, the gene of the direct or indirect inducing cell death of energy when similarly through engineering approaches makes it to be expressed in surface immumoglobulin molecule generation antigen cross-linking with other host cells outside the early stage B cell lymphoma cell, described necrocytosis is independent of the sequencing apoptosis, in the early stage B cell lymphoma clone the when latter occurs in antigen cross-linking.

In above chosen process, can adopt multiple matrix to come antigen-presenting and crosslinked special membrane immunoglobulin acceptor.They include, but are not limited to magnetic bead, the tissue culturing plate of coating protein matter, and transfection the cell of coding purpose antigenic gene.The example that can transfection be used to efficiently express the antigenic cell of purpose includes, but are not limited to L cell and NIH 3T3 cell.But,, then be necessary at first to remove the host cell that any among the host cell group who expresses immunoglobulin (Ig) can express the antibody that the membrane antigen with non-transfected cells responds if recombinant chou antigen is expressed and presented to the utilization cells transfected.More this removal can or be taken turns to be adsorbed onto on the non-transfected cells that is fixed on the solid substrate and realize by one.Just might utilize the transfectant of antigen expressed just selecting to express the cell of special recombinant antibody then.In a preferred embodiment, the negative, positive that repeats alternate cycles is selected, up to the concentration effect that reaches expection.

In just selecting an embodiment of step, the bone-marrow-derived lymphocyte oncocyte of expressing antibodies is adhered on the solid substrate, combined the anti-CD19 and/or the anti-CD20 antibodies of B cell-specific on this matrix.Induce the adhesion indicator cells that the hydrolysis phenomenon takes place,, comprise that all virus immunity sphaeroprotein heavy chain recombinant chous are discharged in the nutrient solution with kytoplasm content with them.The cell that reclaims in nutrient solution of results and the recombinant virus of cell debris are carried out enrichment, and those can give enrichment selecting the recombinant virus of antigenic specific heavy chain immunoglobulin when being coded in some also not certified light chain combination.Newly having infected this enrichment recombinant virus group and having carried out several in addition selections that antigen drives of taking turns in the unselected initial plasmid group cells transfected with the same various light chains of coding subsequently, cause further enrichment purpose heavy chain.After repeating this chosen process several times, be separated to a small amount of heavy chain, these heavy chains possess best specificity to specific antigen when combining with some unidentified light chain.

Can give required specific light chain when combining with the previous heavy chain of selecting in order to choose, by being structured in based on the light chain recombinant chou library in the carrier of bovine vaccine and coming host cells infected with MOI=1, usefulness, the plasmid recombinant chou of one of heavy chain of the previous selection of usefulness subsequently carries out transfection and repeats aforesaid whole chosen process.The chosen process of carrying out the above-mentioned antigen driving of several cycles can be separated to the best light chain partner of this heavy chain.

In a further preferred embodiment, utilization has been carried out similar strategy at the binding characteristic of the cell of film surface expression specific antibody.This strategy is set forth as Fig. 5; do not use the early stage B cell lymphoma that the crosslinked generation apoptosis of acceptor is replied as indicator cells; but by be attached to coupling antigenic synthetic particle or polymkeric substance, the specific host cell of purpose immunoglobulin (Ig) is selected to express in the surface that perhaps is attached to the transfectional cell of expressing specific antigen.In this situation, indicator cells is because the ability of its high level expression membrane immunoglobulin acceptor, but not the crosslinked generation apoptosis of membrane immunoglobulin acceptor is replied and selected.Preferred clone comprises the negative plasmoma of immunoglobulin (Ig).With the specificity of chosen process, the background other problems relevant with efficient handled according to above description.

Embodiment 3

From 10 ⁹Select to have specific specific antibody in the library of kind heavy chain immunoglobulin and light chain combination.

The avidity that can be selected from the specific antibody in a library is the function of this library size.Usually, the number of the heavy chain of library representative and light chain combination is big more, exists and to choose the possibility of high-affinity antibody just big more.The previous work prompting of adopting the phage display method to carry out, for many antigens, one comprises 10 ⁹The library size of planting the combination of heavy chain immunoglobulin and light chain is enough chosen the specific antibody of comparison high affinity.In theory, might make up one and have 10 ⁹The library of individual recombinant chou, wherein each recombinant chou is expressed a kind of heavy chain of uniqueness and a kind of light chain of uniqueness, perhaps has the strand construct in the combination site that has comprised heavy chain and variable region of light chain.But most preferred method is by making up 10 ⁵Individual heavy chain immunoglobulin and 10 ⁴Two libraries of individual light chain immunoglobulin, they can coexpression go out all 10 ⁹Plant possible combination, thereby produce the antibody combination of this number.In the present embodiment, there is bigger diversity in the heavy chain pond, than relevant light chain the specific antigen binding site is played bigger effect because often find heavy chain.

3.1 heavy chain gene.By minimum 10 ⁵Individual heavy chain immunoglobulin cDNA transferring plasmid recombinant construction titre is about 10 ⁶Bovine vaccine recombinant chou library, plasmid recombinant chou wherein is the RNA synthetic that comes from 100 bone marrow donor ponds by previously described method (embodiment 1) origin.As mentioned below, this library must be expanded to titre and be at least 10 ⁹Individual heavy chain recombinant chou.The method in a preferred amplification library is with having 10 ³The single pond of individual bovine vaccine heavy chain recombinant chou infects about 5 * 10 ⁴Little culture of individual BSC1 cell.Usually through after 48 hours the infection, virus titer can increase more than 1000 times.A plurality of concentrate separately the amplicon virus titres to reduce since competitive subgroup growth phase to rapid danger of losing the recombinant chou subgroup.

3.2 light chain gene.By minimum 10 ⁴Individual light chain immunoglobulin cDNA transferring plasmid recombinant construction titre is about 10 ⁵Bovine vaccine recombinant chou library, plasmid recombinant chou wherein is the RNA synthetic that comes from the bone marrow donor pond as origin as described in the embodiment 1.Select for the many wheels that are used for heavy chain described below, this library must be expanded to titre is 10 ¹⁰To 10 ¹¹Individual light chain recombinant chou.The method in a preferred amplification library is with having 10 ³The single pond of individual bovine vaccine light chain recombinant chou infects about 5 * 10 ⁴100 parts of little cultures of individual BSC1 cell.To further increase from the virus recombinant of each recovery these 100 infected cultures is that titre is greatly about 10 ⁸To 10 ⁹The independent pond of individual virus recombinant.For simplicity, these light chain ponds are labeled as L1 to L100.

3.3 select the heavy chain immunoglobulin recombinant chou.To 100 part 10 ⁷Individual non-generation myeloma cell's culture, preferred Sp2/0, or early stage B cell lymphoma, preferred CH33, infect with the bovine vaccine heavy chain recombinant chou of living with MOI=1, infect the bovine vaccine light chain recombinant chou (seeing below) of MOI=1 to 10 simultaneously through psoralene (4 '-aminomethyl Trisoralen) deactivation.In order to carry out the psoralene deactivation, with the psoralene of 10 μ g/ml with 10 ⁸To 10 ⁹The acellular virus of pfu/ml was handled 10 minutes at 25 ℃, was exposed to long wave UV line (365-nm) 2 minutes (Tsung, K., J.H.Yim, W.Marti, R.M.L.Buller, and J.A.Norton.J.Virol.70:165-171 (1996)) then.The virus of handling through psoralene is reproducible not, but can express early stage virogene, comprises being in early days but not being the recombination of viral promotors in late period under regulating and control.Under these conditions, can be assembled into immunoglobulin molecules with single heavy chain of planting of expressing in each cells infected by the recombinant chou synthetic light chain of handling through psoralene.

Selection still is that MOI=10 uses the light chain recombinant chou through the psoralene deactivation to infect and can be combined in a relative concentration in the positive cell by the specific H+L chain of influence with MOI=1, i.e. height when MOI=1, lower when MOI=10 (because being diluted) by a plurality of light chains.Specific immune sphaeroprotein concentration is low on the cell surface, and correspondingly density descends, and can select the antibody high to the purpose ligand affinity.On the other hand, estimate that the special of high density conducted through the combination or the signal of immunoglobulin receptor mediation by the physical efficiency promotion.

Combination of describing by embodiment 2 or signal conduction are carried out after first round antigen-specific selects, from each culture, reclaim the enriched populations of recombinant virus, its titre may be to add 1% to 10% of viral titre in this primary election process, depends on the background level of the spontaneous release of non-specific binding or virus.For simplicity, the heavy chain recombinant chou pond of reclaiming in will selecting the first round is labeled as Hla to H100a, and described pond has obtained respectively from the virus through psoralene processing of initial light chain recombinant chou pond L1 to L100.

Take turns selection in order under the condition identical, to carry out second, still be necessary 10 to 100 times of the titre amplifications of the heavy chain recombinant chou that will be recovered to the first round.Take turns selection for second, infect non-generation myelomatosis or early stage B cell lymphoma with the viral heavy chain recombinant chou of living with through the light chain recombinant chou that psoralene was handled again, thereby, for example use the heavy chain recombinant chou that from the H37a of pond, reclaims and infect 10 from the light chain recombinant chou of handling through psoralene in the initial L37 pond that is used to select H37a ⁷The identical culture of individual cell.For simplicity, take turns the heavy chain recombinant chou that reclaims from the H37a pond in the selection with second and be labeled as H37b etc.

Second take turns selection after, special virus recombinant compare with the viral colony of beginning usually may be by enrichment 10 times or more than.In this case, need not carry out third round under the identical condition and select taking turns, even because under low 10 times titre, special clone also can obtain fine embodiment with the first round or second.Therefore select for third round, again with the viral heavy chain recombinant chou of living with from the light chain recombinant chou infection of handling through psoralene 10 of identical tanks ⁶100 parts of cultures of individual non-generation myelomatosis or early stage B cell lymphoma cell.The 5th take turns selection after, the quantity of cells infected can reduce 10 times again.

3.4 identify antigen-specific heavy chain recombinant chou.

(a) every take turns selection after, can determine in the particular pool (for example H37f) the antigen-specific heavy chain whether be enriched to 10% or more than, this can choose 10 single virus pfu and detect antigen-specific when linking to each other with the light chain in original L37 pond by concentrating from this heavy chain.Because light chain colony comprise be distributed in 100 concentrate separately 10 ⁴Individual different cDNA, each pond on average has about 10 ²Individual different light chain.Even selected heavy chain only just giving required antigen-specific with can combine for a certain class light chain that the light chain that utilizes is concentrated the time, still has 1% cell that has infected the pond of light chain at random of selected heavy chain recombinant chou and MOI=1 can express required specificity.If cell is to use the light chain of MOI=10 to infect, this frequency can bring up to average 10%.The specific method of preferred confirmation is to infect the early stage B cell lymphoma clone of CH33 with heavy chain immunoglobulin and light chain pond, the transfection of this clone the reporter constructs of easy detection, for example can be by the luciferin that promotor drove of the crosslinked activated CH33 of membrane receptor gene by BAX or another.If the heavy chain that chooses with these 100 kinds of concentrating or more light chains in any linking to each other the time can give required antigen-specific, then with the heavy chain recombinant chou of plaque purification with can the easy detected signal of generation after this transfectant is infected in relevant light chain pond.Notice that this identical method can be used to analyze heavy chain, no matter they are that the specific combination or the conduction of special signal of the immunoglobulin receptor mediation by infected cell selected.

(b) alternative method of identifying the most promising antigen-specific heavy chain be screening in selected colony most representative those.Can utilize the carrier special primer that inserts the site side to separate and insert fragment, and these can be inserted the frequency that sequencing fragment is determined viewed sequence by pcr amplification.But, in this situation, still need to resemble and identify relevant light chain described below.

3.5 select the light chain immunoglobulin recombinant chou.In case be separated to the antigen-specific heavy chain, can be as when the concentrated separation that is used to select heavy chain combines with this heavy chain, giving the light chain of antigen-specific as described in 3.4 (a).Perhaps, also can from bigger library, be chosen in another light chain that can strengthen avidity when combining with same heavy chain.For this purpose, by minimum 10 ⁵Individual light chain immunoglobulin cDNA transferring plasmid recombinant construction titre is approximately 10 ⁶Bovine vaccine recombinant chou library, wherein the transferring plasmid recombinant chou is by previously described method (embodiment 1) synthetic.The program of describing in 3.3 is put upside down, and the single specificity heavy chain recombinant chou of handling with the live virus light chain recombinant chou of MOI=1 and the psoralene that chooses infects non-generation myelomatosis or B cell lymphoma in early days now.In order to promote to choose the more immunoglobulin (Ig) of high-affinity, preferably infect and dilute the right concentration of each special H+L chain by light chain with MOI=10.

3.6 under the situation that has single light chain immunoglobulin to exist, select the heavy chain immunoglobulin recombinant chou.If identified a candidate light chain, then can simplify chosen process to the contributive heavy chain immunoglobulin of specific antibodies specificity.Just may be so when for example before having chosen a mouse monoclonal antibody.The mouse variable region of light chain can be transplanted to the pairing of optimizing on people's constant region of light chain with people's heavy chain, adopted the researchist of phage display method to be called " directiveness is selected " (Jespers by other before this process, L.S., A.Roberts, S.M.Mahler, G.Winter, H.R. and Hoogenboom.Bio/Technology 12:899-903,1994; Figini, M., L.Obici, D.Mezzanzanica, A.Griffiths, M.I.Colnaghi, G.Winters and S.Canevari.Cancer Res.58:991-996,1998).In theory, if people's variable region gene framework region also is transplanted on the mouse light chain variable region sequence, then can further carry out this molecule coupling (Rader, C., D.A.Cheresh, and C.F.Barbas, Proc.Natl.Acad.Sci.USA 95:8910-8915).Choose with this adorned antigen-specific light chain paired anyone heavy chain self can be as from more diversified basis of concentrating as the beautiful woman's light chain of selection as described in 3.5.

Embodiment 4

From being structured in adenovirus, human antibodies specific is selected in the cDNA library in simplexvirus or the retroviral vector

4.1 simplexvirus.Produce existing describe (T.A.Stavropoulos, the C.A.Strathdee.1998 J.Virology 72:7137-7143) of method of the non-auxiliary virus stoste of the infectious type simple herpesvirus amplicon of reorganization (HerpesSimplex Virus Amplicons).Utilize this method, be structured in the human immunoglobulin heavy chain in the sub-carrier of plasmid amplification and/or the cDNA library of light chain gene or its fragment (comprising single-chain fragment) and might be packaged into infectious amplicon particle library.Another amplicon library that utilizes the amplicon library of immunoglobulin heavy chain gene structure and utilize light chain immunoglobulin gene to make up can the non-generation myeloma cell line of coinfection.Combine with purpose is antigenic by selection, can enrichment can express myeloma cell with required specific immunoglobulin gene combination.The simplexvirus amplicon can be in cells infected express transgenic stably.The cell of choosing at associativity in the first round will keep their immunoglobulin gene combination, and can stably express and have this species specific antibody.This makes and can repeat to select circulation to have required specific immunoglobulin gene up to being separated to.Can also attempt causing the selection strategy of necrocytosis.The amplicon vector that reclaims from the selected cell of those death can not be used to infect fresh target cell, because do not having under the situation of helper virus, these amplicons are replication defectives, can not be packaged into infectious form.Amplicon vector contains the replication origin and the antibiotics resistance gene of plasmid.This makes to be transformed into by DNA that will purifying obtains from selected cell and reclaims selected amplicon vector in the bacterium.Select just can be separated to the bacterial cell that has transformed this amplicon vector with suitable microbiotic.If heavy chain is used different microbiotic (for example ammonia benzyl and kantlex) resistant genes with the light chain amplicon vector, then can from identical selected cell mass, choose heavy chain and light chain gene respectively.Can from bacterium, extract the amplicon plasmid DNA, and by with the HSV genomic dna cotransfection packing cell of packing defective it being packaged into infectious viral particle with amplicon DNA.Can gather in the crops infectious amplicon particle then, infect fresh target cell group with it and carry out another and take turns selection.

4.2 adenovirus.Existing (S.Miyake, M.Makimura, Y.Kanegae, S.Harada, Y.Sato, K.Takamori, C.Tokuda, the I.Saito.1996 Proc.Natl.Acad.Sci.USA 93:1320-1324 of describing of method for preparing recombinant adenovirus; T.C.He, S.Zhou, L.T.Da Costa, J.Yu, K.W.Kinzler, B.Volgelstein.1998 Proc.Natl.Acad.Sci.USA 95:2509-2514).Utilize one of these methods, can be in adenovirus carrier the construction cDNA library.E3 or E4 district that cDNA inserts adenovirus will produce reproducible recombinant virus.This library can be used for similar application, such as make up bovine vaccine cDNA library by three molecular recombination.For example heavy chain cDNA library can be inserted the E3 or the E4 district of adenovirus.This will produce reproducible heavy chain library.The light chain cdna library can be inserted the E1 gene of adenovirus, produce the replication defective library.By infecting the trans cell that adenovirus E 1 is provided of energy, such as 293 cells, this replication defect type light chain library of can increasing.These two libraries can be used for the use reproducible bovine vaccine heavy chain library described with through the similar selection strategy in the bovine vaccine light chain library of psoralene deactivation.

4.3 the advantage of vaccinia virus.Vaccinia virus is having some advantages than simplexvirus or adenovirus aspect the construction cDNA library.At first, vaccinia virus is duplicated in the kytoplasm of host cell, and HSV and adenovirus are duplicated in nuclear.For vaccinia virus, frequency ratio HSV that the cDNA recombinant transfer plasmid is recombinated in kytoplasm or adenovirus be transported in the nuclear and pack/the efficient height of recombinating.The second, vaccinia virus can be with not sequence-dependent mode plasmid replication, and adenovirus or simplexvirus can not (M.Merchlinsky, B.Moss.1988 CancerCells 6:87-93).CDNA recombinant chou transferring plasmid duplicates the frequency that may cause producing recombinant virus in bovine vaccine higher.Though we have described the possibility in construction cDNA library in simplexvirus or adenovirus carrier, should emphasize also not report and utilize these methods construction cDNA library in these virus vector.

4.4 retrovirus.Existing (T.Kitamura, M.Onishi, S.Kinoshita, A.Shibuya, A. Miyajima and the G.P.Nolan.1995 PNAS 92:9146-9150 of describing in construction cDNA library in the retroviral vector of replication defective; I.Whitehead, H.Kirk and R.Kay.1995 Molecular and CellularBiology15:704-710.).Integrate behind the retroviral vector target cell infection, the target cell and can induce stable transgene expression because they can be transduceed effectively, retroviral vector is widely used.Another retrovirus library that utilizes the retrovirus cDNA library of immunoglobulin heavy chain gene structure and make up with light chain immunoglobulin gene can be used for the non-generation myeloma cell line of coinfection.Combine with purpose is antigenic by selecting, can have the myeloma cell that required specific immunoglobulin gene makes up to expression and carry out enrichment.Make up by keeping its immunoglobulin gene in the first round, and stably expression has this specific immunoglobulin (Ig) in conjunction with the cell of choosing.This makes that can repeat this selection circulation has required specific immunoglobulin gene until being separated to.

Embodiment 5

Three molecular recombination

5.1 the preparation of expression library.Present embodiment has been described a kind of three molecular recombination methods, and this method is utilized the vaccinia virus vector of modified and relevant transferring plasmid, produces the recombinant chou vaccinia virus near 100%, and made up the representative DNA library first effectively in vaccinia virus.Fig. 6 has illustrated this three molecular recombination method.

5.2 the structure of carrier.Previously described vaccinia virus transferring plasmid pJ/K (derived from the plasmid of pUC13) carries the vaccinia virus thymidine kinase gene (Merchlinsky that contains NotI site in the frame, M.et al., Virology 190:522-526), it is further modified to be with a last strong vaccinia virus promotor, follow by Not I and Apa I restriction site.Two different carriers p7.5/tk and pELJtk comprise respectively 7.5K vaccinia virus promotor or strong synthetic early stage/(E/L) promotor in late period (Fig. 7).Front, Apa I site is a strong translation initiation sequence that comprises the ATG codon.Import this and modify in vaccinia virus thymidine kinase (tk) gene, making its side is the regulation and control and the encoding sequence of viral tk gene.The intragenic modification of tk is transferred in the genome deutero-vNotI-carrier of vaccinia virus WR strain by the homologous recombination of side tk sequence in these two novel plasmid carriers, produces new virus carrier v7.5/tk and vEL/tk.Importantly, after these virus vector are carried out Not I and Apa I digestion with restriction enzyme, separate two big viral dna fragments, they respectively contain the independent non-homogeneous section of bovine vaccine tk gene, then comprise needed all genes of assembling infectious viral particle together.Embodiment 1 has described other details about the structure of these carriers and evaluation, and they directly connect other application of dna fragmentation in vaccinia virus.

5.3 improve the frequency of vaccinia virus recombinant chou.The ordinary method of preparation recombinant chou has been utilized the homologous recombination between recombinant chou bovine vaccine transferring plasmid and the viral genome in vaccinia virus.Table 3 has shown the result of a model experiment, and the frequency that homologous recombination takes place behind the cell that has infected vaccinia virus the transfection of recombinant chou transferring plasmid is measured in this experiment under standard conditions.In order to promote Function detection, insert coding and H-2K in the NotI site of transferring plasmid tk gene ^bThe minigene of the ovalbumin immundominance 257-264 peptide epitopes that connects together.The result of homologous recombination is that the tk gene that is interrupted in all recombinant virus is all replaced by wild-type tk+ gene.This can be used as the mark of reorganization, and is because the tk-people 143B cell that has infected tk-virus has resistance to the toxicity of BrdU, different with the cell that has infected wild-type tk+ virus.Can come by the viral pfu on the 143B cell of cultivating under the situation that 125mM BrdU is arranged to give a mark to recombinant virus.

The frequency of the recombinant chou that obtains by this way is 0.1% order of magnitude (table 3).

Table 3: produce the recombinant chou vaccinia virus by conventional homologous recombination
					Virus *	DNA	Titre w/o BrdU	Full scale w/BrdU	% recombinant chou * *
Bovine vaccine	---	4.6×10 7	3.0×10 3	0.006
					Bovine vaccine	30ng pE/Lova	3.7×10 7	3.2×10 4	0.086
Bovine vaccine	300ng pE/Lova	2.7×10 7	1.5×10 4	0.056

* vaccinia virus strain vNotI

* % recombinant chou=(titre when BrdU is arranged/when not having BrdU titre) * 100

Such recombination frequency is too low, effectively construction cDNA library in the bovine vaccine carrier.Utilize following two steps to produce the vaccinia virus recombinant chou of higher frequency.

(1) the plasmid transfer vector transfection is behind the cell that has infected vaccinia virus, and a limiting factor that produces the frequency of virus recombinant by homologous recombination is, virus infection efficient is very high, and the plasmid DNA transfection relative efficiency is low.The many cells infecteds of result do not absorb plasmid recombinant, therefore can only produce wild-type virus.In order to reduce dilution to recombination efficiency, with the mixture transfection of naked virus DNA and plasmid recombinant DNA in the mammalian cell that has infected fowlpox virus (FPV).(the Scheiflinger that described in the past as other researchists, F. etc., 1992, Proc.Natl.Acad.Sci.USA 89:9977-9981), FPV does not duplicate in mammalian cell, but can be for transfection the cell of non-infectious exposed bovine vaccine DNA provide the ripe vaccinia virus particle of packing necessary subsidiary function.Only this modification to homologous recombination technique just can improve the frequency of virus recombinant about 35 times, reaches 3.5% (table 4).

Table 4: produce the recombinant chou vaccinia virus by the homologous recombination of modifying.

Virus	DNA	Titre w/o BrdU	Titre w/BrdU	The % recombinant chou
					PFV	Do not have	0	0	0
Do not have	Bovine vaccine WR	0	0	0
					PFV	Bovine vaccine WR	8.9×10 6	2.0×10 2	0.002
PFV	Bovine vaccine WR+pE/Lova (1: 1)	5.3×10 6	1.2×10 5	2.264
					PFV	Bovine vaccine WR+pE/Lova (1: 10)	8.4×10 5	3.0×10 4	3.571

* % recombinant chou=(titre when BrdU is arranged/when not having BrdU titre) * 100

Table 4. is paved with BSC1 cell (5 * 10 with the fowlpox virus strain HP1 infection individual layer of MOI=1.0 ⁵Cells/well).After two hours, inhale and to remove supernatant, cell is washed twice with the Opti-MemI substratum, through lipofectamine with 600ng bovine vaccine strain WR genomic dna independent or with 1: 1 or (bovine vaccine: the plasmid pE/Lova transfection of molar ratio plasmid) in 1: 10.This plasmid contains the fragment of the coding SIINFEKL epi-position of ovalbumin cDNA, known this epi-position and mouse I class MHC molecule K ^bThe high-affinity combination.The expression of this minigene is subjected to strong synthetic property bovine vaccine in a morning/late period promoter regulation.This insertion fragment side is bovine vaccine tk DNA.Harvested cell after three days freezes/melts three-wheel and extracts virus in ℃ water-bath of dry ice primary isoamyl alcohol/37.By on people TK-143B cell, having and carrying out plaque when not having BrdU and detect the titre of measuring thick virus stock solution used.

(2) by infected the cell of FPV with the big mixture transfection of plasmid recombinant and two near the vaccinia virus v7.5/tk dna fragmentation of 80kb and 100kb, make the frequency of virus recombinant that another tangible raising arranged, wherein said two fragments produce with Not I and Apa I digestion with restriction enzyme.Because imported Not I and Apa I site in the tk gene, each all comprises a fragment of tk gene these big bovine vaccine DNA arms.Owing to do not have homology between these two tk gene fragments, the unique method that these two bovine vaccine arms can be coupled together is to come bridge joint by the homology tk sequence of inserting the fragment side in the recombinant chou transferring plasmid.The result of table 5 shows, determines by the BrdU resistance of infected tk-cell, and the infectious vaccinia virus that produces in the triple turn transfect cell DNA that recombinated more than 99% inserts fragment.

Table 5: utilize three molecular recombination to produce 100% recombinant chou vaccinia virus.

Virus	DNA	Titre w/o BrdU	Full scale w/BrdU	% recombinant chou *
					PFV	The v7.5/tk that does not have cutting	2.5×10 6	6.0×10 3	0.24
PFV	NotI/Apal v7.5/tk arm	2.0×10 2	0	0
					PFV	NotI/Apal v7.5/tk arm+pE/Lova (1: 1)	6.8×10 4	7.4×10 4	100

* % recombinant chou=(titre when BrdU is arranged/when not having BrdU titre) * 100

Table 5. genomic dna (1.2 microgram) of ApaI and NotI digestion with restriction enzyme bovine vaccine strain V7.5/tk.The DNA that digestion is good is divided into two parts.It is a that (bovine vaccine: plasmid) molar ratio mixed with pE/Lova with 1: 1.This plasmid contains the fragment of the coding SIINFEKL epi-position of ovalbumin cDNA, known this epi-position and mouse I class MHC molecule K ^bThe high-affinity combination.The expression of this minigene is subjected to strong synthetic morning/bovine vaccine in a late period promoter regulation.This insertion fragment side is bovine vaccine tk DNA.With lipofectamine with the DNA transfection in the individual layer BSC1 cell that is paved with (5 * 10 ⁵Cells/well), this cell has infected two hours with the FPV of MOI=1.0 earlier.The untreated genome V7.5/tk of the 600ng DNA transfection of one duplicate samples.Harvested cell after three days freezes/melts three-wheel and extracts virus in ℃ water-bath of dry ice primary isoamyl alcohol/37.By on the TK-143B cell, having and carrying out plaque when not having BrdU to select and detect the titre of measuring thick virus stock solution used.

5.4 in vaccinia virus, make up representative cDNA library.The construction cDNA library shows that known cell mRNA sequence is expressed typically in the bovine vaccine carrier.In p7.5/tk transferring plasmid and v7.5/tk virus vector, import other modification, so that improve the efficient that recombinant chou is expressed in the cells infected.Described modification is included in three different frames and imports translation initiation site, imports translation and transcription termination signal and is used for other restriction sites that DNA inserts.

At first, with the HindIII site of HindIII J fragment (bovine vaccine tk gene) of p7.5/tk, produce pBS.Vtk from this plasmid subclones to pBS phagemid (Stratagene).

Secondly,, remove the part of the original multiple clone site of this plasmid, handle, reconnect, produce pBS.Vtk.MCS-with mung-bean nuclease with SmaI and PstI digestion pBS.Vtk.This treating processes has been removed the SmaI among the pBS.Vtk, BamHI, SalI and PstI single endonuclease digestion site.

Once more, the purpose here is to import new multiple clone site in the downstream of the 7.5k of pBS.Vtk.MCS-promotor.This new multiple clone site produces through PCR by utilizing 4 different upstream primers and a shared downstream primer.These 4 PCR products will not conform to the ATG initiator codon or will contain the ATG initiator codon that is in three each in may frames.In addition, each PCR product contains translation stop codon of three kinds of frames at 3 ' end, and vaccinia virus is transcribed dual termination signal.These 4 PCR products are connected respectively to the N0tI/ApaI site of pBS.Vtk.MCS-, produce 4 carriers, p7.5/ATGO/tk, p7.5/ATG1/tk, p7.5/ATG2/tk, and p7.5/ATG3/tk, Figure 12 have shown their sequence modifications with respect to p7.5/tk.Each carrier includes BamHI, SmaI, PstI and SalI single endonuclease digestion site, be used for cloned DNA and insert fragment, they or utilize self endogenous translation initiation site (in the p7.5/ATGO/tk carrier), perhaps utilize the carrier translation initiation site (p7.5/ATGl/tk, p7.5/ATG3/tk and p7.5/ATG4/tk) that is in one of three kinds of possibility frames.

In a model experiment,, and be connected in four adorned p7.5/tk transferring plasmids each by the synthetic cDNA of the poly-A+mRNA of mouse tumor cell line (BCA39).By prokaryotic host cell such as intestinal bacteria in the additive method as described herein or known in the art transferring plasmid that increases that goes down to posterity.With 20 micrograms through the v/tk vaccinia virus DNA arm of Not I and Apa I digestion and four kinds of plasmid recombinant cDNA libraries etc. the molar mixture transfection in the BSC-1 cell that infects the FPV helper virus, carry out three molecular recombination.The total titre of the virus of gathering in the crops is 6 * 10 ⁶Pfu wherein is the BrdU resistance more than 90%.

In order to determine that cDNA inserts segmental size distribution in the recombinant chou bovine vaccine library, choose one separation plaque with aseptic pasteur transfer pipet, transfer in the 1.5ml test tube that contains 100ul phosphate buffered saline buffer (PBS).By in dry ice/primary isoamyl alcohol, freezing/melt three-wheel virus is discharged from cell in 37 ℃.Approximately the tk-people 143B cell in the 250ul final volume is wherein contained in 1/3rd holes that are used for infecting 12 orifice plates of each viral plaque.Two hours period of infection last, the DEME (DEME-2.5) and the BudR that contain 2.5% foetal calf serum with 1ml cover each hole, and the consumption of Brdll is enough to make final concentration to reach 125ug/ml.With cell in 37 ℃ at CO ₂Insulation is 3 days in the incubator.The 3rd day harvested cell, centrifugation is resuspended among the 500 μ l PBS.Freeze/melt circulation releasing virus from cell through three-wheel as mentioned above.Infect the BSC-1 monolayer cell that is paved with in the 50mm tissue culture ware with 20% of every part of viral liquid storage, final volume is 3ml DEME-2.5.2 hours period of infection last covers cell with 3ml DMEM-2.5.With cell at CO ₂Be incubated 3 days in 37C in the incubator.The 3rd day harvested cell, centrifugation is resuspended in 300 μ l PBS.Freeze/melt circulation through three-wheel as mentioned above and discharge virus in the cell.The thick viral liquid storage of 100 μ l is transferred in the 1.5ml test tube, added equal-volume and melt 2% good low melting-point agarose, virus/agarose mixture is transferred in the pulsed field gel sample cell.After agar solidifies they are taken off from sample cell, be cut into equal three parts.Whole three parts are all transferred in the same 1.5ml test tube, add 250 μ l0.5M EDTA, 1% Sarkosyl and 0.5mg/ml Proteinase Ks.Adhesive tape is incubated 24 hours in 37 ℃ in solution.In 500 μ l 0.5X tbe buffer liquid, wash several times, the part of each blob of viscose is transferred in the hole of 1% low melting-point agarose gel.After adding adhesive tape, melt 1% good low melting-point agarose by other interpolation the hole is sealed.Then with gel in Bio-Rad pulsed field gel electrophoresis device in 200 volts, 8 pulse per second (PPS) times, electrophoresis is 16 hours in 0.5X TBE.Glue is dyeed in EB, downcut the agarose part that contains the bovine vaccine genomic dna, transfer in the 1.5ml test tube from glue.With β-gelase (Gibco) according to manufacturer's recommendation purifying bovine vaccine DNA from agarose.The bovine vaccine DNA that purifying is good is resuspended in 50ul ddH ₂O.Each 1 μ l of DNA liquid storage is as the template of polymerase chain reaction (PCR), and PCR uses bovine vaccine TK special primer MM428 and MM430 (at the side that inserts the site) and Klentaq polysaccharase (Clontech) to carry out according to being recommended in the 20 μ l final volume of manufacturers.Reaction conditions comprises 95 ℃ of initial denaturing steps of 5 minutes, is 94 ℃ of 30 round-robin 30 seconds then, 55 ℃ 30 seconds, 68 ℃ 3 minutes.Each 2.5 μ l of PCR reaction system separate on 1% sepharose, dye with EB.Observe the amplified fragments of different sizes.After proofreading and correct at the side carrier sequence that increases among the PCR, insert segmental size between 300 to 2500bp.

The representativeness of gene product express to be that frequency that frequency by showing special cDNA recombinant chou in the bovine vaccine library and same cDNA recombinant chou occur is as broad as long and to set up in this library in the standard plasmid library.Be that example has illustrated this point with the IAP sequence in table 6, this sequence before had been presented in the mouse tumour and had been raised.By the little culture that infects the 143Btk-cell under the situation that has BDUR to exist 20 the independent ponds from the bovine vaccine library of increasing, wherein each on average has 800 or 200 viral pfu.From each infected culture, extract DNA after 3 days, with endogenous retrovirus (IAP, the intracisternal A particle) sequence of sequence specific primers evaluation before whether the PCR detection exists.The Poisson analysis revealed frequency that positive pond frequency is carried out has an IAP recombinant chou (table 6) for about per 500 viral pfu.Similarly, will increase in independent pond from 20 of plasmid library by transforming DH5 α bacterium, there are average 1400 or 275 bacterium cfu in described pond.Detection sees whether there is identical IAP sequence from the plasmid DNA in each pond.The Poisson analysis display frequency that positive pond is carried out is that per 450 plasmids have an IAP recombinant chou (table 6).

The limiting dilution analysis of the IAP sequence in table 6 recombinant chou bovine vaccine library and the conventional plasmid cDNA library
						The positive hole of PCR#	F 0	μ	Frequency
The #PFU/ hole		The bovine vaccine library
					800	18/20	0.05	2.3	1/350
200	6/20	0.7	0.36	1/560
					The #PFU/ hole		Plasmid library
1400	20/20	0	-	-
					275	9/20	0.55	0.6	1/450

F ₀The ratio of=negative hole; μ=DNA precursor/hole=-lnF ₀

Similar analysis has been carried out in displaying in the bovine vaccine library to α tubulin sequence, has obtained similar result.The frequency of the sequence of Xuan Zeing in two libraries that made up by identical tumour cDNA is suitable at random, though this explanation structure bovine vaccine library is more complicated than making up plasmid library, and does not resemble latter's routine so certainly, can represent tumour cDNA sequence equally.

Discuss

Above-described three molecular recombination strategies can produce 100% virus recombinant nearly.This is significantly improved than the method that at present prepares virus recombinant by the cell that has infected vaccinia virus with the plasmid transfer vector transfection.The frequency that a kind of method in back produces virus recombinant only has 0.1% order of magnitude.The high yield of virus recombinant makes and can make up genome or cDNA library efficiently for the first time in the vaccinia virus derivative vector in three molecular recombination.In first batch of experiment, the mixture of the bovine vaccine vector arms that digested with 20 microgram NotI and ApaI with etc. volumetric molar concentration tumour cell cDNA together after the transfection, obtaining titre is 6 * 10 ⁶Recombinant virus.New, effectively screening and the selection strategy that separates specific gene group and cDNA clone created in this technical progress.

The three molecular recombination methods of describing in the literary composition can be used for other viruses, such as mammalian virus, comprise bovine vaccine and simplexvirus.Usually produce two viral arms that do not have homology.The unique channel that these two viral arms can link up is to come bridge joint by inserting segmental side homologous sequence in the transfer vector (such as plasmid).When two viral arms and transfer vector were present in same cell, unique infectious virus of generation was that the DNA in the transfer vector inserts segmental recombinant chou.

The library that in bovine vaccine and other mammalian virus, makes up by three molecular recombination methods of the present invention have with literary composition in identify at vaccinia virus and in CTL screening system of the present invention that purposes in the purpose antigen is described confers similar advantages arranged.Estimate to be structured in confers similar advantages is arranged when more complicated analysis is carried out in DNA library in bovine vaccine or other mammalian virus in eukaryotic cell.This alanysis includes, but are not limited to screen the DNA of encode true karyocyte acceptor and part.

Embodiment 6

The preparation of transferring plasmid

Can prepare the transfer vector that is used to clone by known method.Preferable methods comprises with suitable restriction enzyme (for example SmaI, SalI or BamHI and SalI) in suitable damping fluid, in suitable temperature with 1-5 microgram carrier cutting at least 2 hours.Digested the carrier that good carrier separates linear digestion through 0.8% sepharose by electrophoresis.Linear plasmid is scaled off from glue, utilize known method purifying from the agarose.

Connect.Utilize known method that cDNA and the good transfer vector of digestion are linked together.In a preferred method, with the T4 dna ligase 50-100ng transfer vector is connected with the cDNA of different concns, use suitable damping fluid to connect 18 to 24 hours at 14 ℃.

Transform.Utilize known method that the sample liquid of ligation is transformed into intestinal bacteria such as among DH10B or the DH5 α by electroporation.The conversion reaction system is taped against on the LB agar plate that contains selection microbiotic (penbritin), 37 ℃ of growths 14-18 hour.All transform bacterias are merged together, use the known method isolated plasmid dna.

Preparation it will be apparent to those skilled in the art that according to the damping fluid of mentioning in the top description to the preferred process of the present invention.

Embodiment 7

Vaccinia virus dna fragmentation and transferring plasmid importing tissue culture cells are carried out three molecular recombination

As described in the embodiment 5 or by other technology known in the art construction cDNA or other libraries in 4 transferring plasmids.Adopt three molecular recombination that this cDNA library is imported vaccinia virus.Infect the BSC1 monolayer cell that is paved with in MOI=1-1.5 with fowlpox virus HP1.Infection is carried out in the serum free medium that is supplemented with 0.1% bovine serum albumin.The BSC1 cell can be placed on 12 holes or 6 orifice plates, 60mm or 100mm tissue culturing plate, or 25cm ², 75cm ², perhaps 150cm ²In the bottle.With the purify DNA of restriction enzyme A paI and NotI digestion from v7.5/tk or vEL/tk.After digestion is finished,, will digest good bovine vaccine arm purifying with centricon 100 pillars with the hot deactivation of enzyme.Between good bovine vaccine DNA of digestion and transferring plasmid cDNA library, form transfection composite then.(Life Technologies Inc.) forms these transfection composites for preferred use Lipofectamine or Lipofectamine Plus.The transfection of carrying out in 12 orifice plates usually needs 0.5 microgram to digest good bovine vaccine DNA and 10ng to the plasmid DNA of 200ng from the library.The transfection of carrying out in bigger culture tube needs the amount of bovine vaccine DNA and transferring plasmid to increase in proportion.After 2 hours, remove fowlpox virus 37 ℃ of infection, add bovine vaccine DNA, transferring plasmid transfection composite.With cell and transfection composite incubation 3 to 5 hours, remove transfection composite afterwards, change to the DMEM that 1ml is supplemented with 2.5% foetal calf serum.With cell at CO ₂Cultivated 3 days in 37 ℃ in the incubator.Harvested cell after 3 days freezes/melts releasing virus by three-wheel dry ice/primary isoamyl alcohol/37 ℃ water-bath.

Embodiment 8

Mammalian cell-infecting

Present embodiment has been described other method with bovine vaccine DNA and transferring plasmid transfectional cell.Can utilize for example following method, carry out three molecular recombination in the host cell by digesting good bovine vaccine DNA and transferring plasmid transfection: calcium phosphate precipitation method (F.L.Graham, A.J.Van derEb (1973) Virology 52:456-467, C.Chen, H.Okayama (1987) Mol.Cell.Biol.7:2745-2752), DEAE-Dextran (D.J.Sussman, G.Milman (1984) Mol.Cell.Biol.4:1641-1643), or electroporation (T.K.Wong, E.Neumann (1982) Biochem.Biophys.Res.Commun.107:584-587, E.Neumann, M.SchaferRidder, Y.Wang, P.H.Hofschneider (1982) EMBO are J.1:841-845).

Embodiment 9

The structure of MVA three molecular recombination carriers

In order to make up modified bovine vaccine Ankara (MVA) carrier that is applicable to three molecular recombination, must in MVA tk gene, insert two single endonuclease digestion restriction endonuclease sites.Complete MVA genome sequence is known (GenBank U94848).The retrieval that this sequence is carried out shows restriction enzyme A scI, RsrII, and SfiI and XmaI do not cut the MVA genome.We have selected restriction enzyme A scI and XmaI, because these two kinds of enzymes have bought easily, and the recognition sequence size of AscI and XmaI is respectively 8bp and 6bp.For these sites being imported MVA tk gene, prepare a construct that contains the reporter gene that both sides are XmaI and AscI site (intestinal bacteria gusA).The Gus gene can obtain from pCRII.Gus (M.Merchlinsky, D.Eckert, E.Smith, M.Zauderer.1997Virology 238:444-451).This report gene construct is cloned in the transferring plasmid, the latter contain bovine vaccine tk DNA flanking sequence and control reporter gene expression the morning/late period the 7.5k promotor.With the Gus special primer by this construct pcr amplification Gus gene.Gus sense strand 5 '

ATGTTACGTCCTGTAGAAACC 3 ' (SEQ ID NO:94) and Gus antisense strand 5 ' TCATTGTTTGCCTCCCTGCTG 3 ' (SEQ ID NO:95).With the Gus special primer this Gus PCR product is done pcr amplification then, wherein said primer is modified, comprises NotI and XmaI site in the adopted primer thereby have, and antisense primer comprises AscI and ApaI site.The sequence of these primers is:

NX-Gus has justice 5 ' AAAGCGGCCGCCCCGGGATGTTACGTCC3 ' (SEQ IDNO:96); With

AA-Gus antisense 5 ' AAAGGGCCCGGCGCGCCTCATTGTTTGCC3 ' (SEQ IDNO:97).

This PCR product digests with NotI and ApaI, is cloned into NotI and the ApaI site of p7.5/tk (M.Merchlinsky, D.Eckert, E.Smith, M.Zauderer.1997 Virology238:444-451).By the homologous recombination of routine, in permission property QT35 or bhk cell, the 7.5k-XmaI-gusA-AscI construct is imported MVA.Through Gus substrate X-Glu (5-bromo-3 indoles-ss-D-glucuronic acid; Clontech) the recombinant chou plaque is selected in (M.W.Carroll, B.Moss.1995 Biotechniques 19:352-355) dyeing.MVA-Gus clone (it also contains XmaI and AscI single endonuclease digestion site) plaque is purified to homogeneous.The large scale culturing thing of amplification MVA-Gus separates naked DNA from the virus of purifying on bhk cell.After XmaI and AscI digestion, MVA-Gus DNA is used for three molecular recombination so that at MVA construction cDNA expression library.

MVA can not finish its life cycle in most mammalian cells.This attenuation will cause longer time ground high level expression recombinant chou cDNAs, but can not be recovered to MVA alive from infected cell.Can't be recovered to MVA alive from the cell of selecting separates the selection repeatedly that the functional cDNA recombinant chou of purpose institute must carry out with overslaugh and circulates.A solution of this problem is to use the helper virus of the host range defective that can compensate MVA to remove to infect the cell that has infected MVA.This helper virus can provide that MVA lacks finishes the necessary gene product of its life cycle.Unlikely the limited helper virus (such as fowlpox virus) of another host range also can compensate the defective of MVA, because these viruses are restricted in mammalian cell equally.The wild-type strain of vaccinia virus should be able to compensate MVA.But in this situation, produce reproducible vaccinia virus and will make selection complicated with the cycle of separating recombinant chou MVA clone.Vaccinia virus that can the working conditions defective, this virus can be provided at and reclaim the needed subsidiary function of MVA of living under the non-permissive condition from mammalian cell, but can not produce reproducible virus.Bovine vaccine D4R open reading frame (orf) coding uracil dna glycosylase.It is necessary that this enzyme is that vaccinia virus is duplicated, and at early expression after the infection (before dna replication dna), it is lethal destroying this gene pairs bovine vaccine.Verified, the mammal cell line of expressing the stable transfection of bovine vaccine D4R gene can compensate D4R defective type vaccinia virus (G.W.Holzer, F.G.Falkner.1997 J.Virology 71:4997-5002).D4R defective type vaccinia virus is a splendid candidate helper virus that can compensate MVA in mammalian cell.

In order to make up D4R compensation clone, utilize primer D4R sense strand 5 ' AAAGGATCCATAATGAATTC AGTGACTGTA TCACACG 3 ' (SEQ ID NO:98) and D4R antisense strand 5 ' CTTGCGGCCG CTTAATAAATAAACCCTTGA GCCC 3 ' (SEQ ID NO:99), from bovine vaccine strain v7.5/tk, clone D4R orf through pcr amplification.The adopted primer that has is wherein comprised the BamHI site by modification, and antisense primer is comprised the NotI site by modification.Pcr amplification also with after BamHI and the NotI digestion, is cloned into D4R orf in BamHI and the NotI site of pIRESHyg (Clontech).This mammalian expression vector contains strong CMV immediate early promoter/enhanser and ECMV internal ribosome entry site (IRES).The transfection of D4RIRESHyg construct in the BSC1 cell, is selected transfected clone with Totomycin.IRES makes that can translate its 5 ' end efficiently contains D4R orf, 3 ' section polycistronic mRNA that contains hygromycin phosphotransferase gene.This will make high-frequency hygromycin resistance clone have required function (these clonal expressions D4R).The BSC1 cell (BSC1.D4R) of expressing D4R can compensate the bovine vaccine of D4R defective type, makes to produce and to breed this defective strain.

In order to make up the bovine vaccine of D4R defective type, from the bovine vaccine genome through the flanking sequence of pcr amplification D4R orf (genomic 100732 to 101388 of bovine vaccine) and 983 bp (5 ' end) and 610bp (3 ' end).Primer D4R side has justice 5 ' ATTGAGCTCTTAATACTTTT GTCGGGTAAC AGAG 3 ' (SEQ ID NO:100), contain SacI (sense strand) and XhoI (antisense) site that is useful on the clone, genomic 99749 to 101998 of the bovine vaccine of can increasing with D4R side antisense 5 ' TTACTCGAGA GTGTCGCAAT TTGGATTTT 3 ' (SEQ ID NO:101).With SacI and the XhoI site of this PCR product cloning, produce pBS.D4R.Flank to pBluescript II KS (Stratagene).The D4R gene contains the EcoRI single endonuclease digestion site of the Nucleotide 3 that originates in 657bp orf, originates in the PstI single endonuclease digestion site of the Nucleotide 433 of this orf.EcoRI and PstI site insertion Gus expression cassette at D4R will be removed most of D4R encoding sequence.Existing people has made up 7.5k promotor-Gus expression vector (M.Merchlinsky, D.Eckert, E.Smith, M.Zauderer.1997 Virology 238:444-451).Utilize primer 7.5 Gus to have justice 5 ' AAAGAATTCC TTTATTGTCATCGGCCAAA 3 ' (SEQ ID NO:102) from this carrier, to separate the 7.5-Gus expression cassette through PCR with 7.5 Gus antisenses, 5 ' AATCTGCAGTCATTGTTTGC CTCCCTGCTG 3 ' (SEQ IDNO:103).Described 7.5Gus has adopted primer to contain the EcoRI site, and the 7.5Gus antisense primer contains the PstI site.Behind the pcr amplification,, and be inserted among the EcoRI and PstI site of pBS.D4R.Flank, produce pBS.D4R-/7.5Gus+ with EcoRI and PstI digestion 7.5Gus molecule.By with pBS.D4R-/7.5Gus+ construct transfection in the BSC1.D4R cell that has infected v7.5/tk, can produce D4R-/Gus+ bovine vaccine through conventional homologous recombination.Separate D4R-/Gus+ virus by on the BSC1.D4R cell, carrying out the plaque purifying with dyeing with X-Glu.This D4R-virus can be used at mammalian cell compensation and rescue MVA genome.

In a relevant embodiment, can use other defect type poxvirus, also can use the wild-type poxvirus of psoralene/UV deactivation in mammalian cell, to save the MVA genome.Psoralene/UV deactivation has been discussed in the literary composition.

Embodiment 10

The structure and the use of D4R three molecular recombination carriers

Poxvirus infection has an obvious suppression effect to host cell proteins matter and RNA are synthetic.These can disturb the selection that those is had the special poxvirus recombinant chou of specific physiological effect to host cell in some cases to the influence that host gene is expressed.Some strain of the crucial early gene defective of vaccinia virus demonstrates host cell proteins matter synthetic inhibition effect is descended greatly.Therefore in the poxvirus vector of certain early gene functional defect, make up the active continuously selection of expressing some recombinant chou of realizing its physiological effect that recombinant chou cDNA library helps relying on some host gene.Destroy the breeding that crucial virogene can stop mutant strain.But trans compensation that can be by host cell or can be provided the function of losing to save replication defect type bovine vaccine strain by the helper virus of this gene of abduction delivering.

Weaken the influence that infection causes host cell signal transduction mechanism, differentiation pathway and transcriptional control greatly with the poxvirus library cells infected group energy that is structured in the replication defective strain.Another important benefits of this strategy is, expresses key gene itself and be exactly the means that a selection directly or indirectly causes the recombinant virus that this transcription regulatory region is activated under the control of target transcription regulatory region.This class example comprises the promotor because of the crosslinked activated gene of surface immumoglobulin acceptor on the early stage B cell precursor; The promotor of the gene of derivative marker after the differentiation of stem cells of perhaps encoding.If the expression of the crucial virogene of such promoters driven has only those directly or indirectly to activate the virus recombinant reproducible of the expression of this transcriptional control so, and is packaged into infectious particles.This method might produce than the much lower background of system of selection based on the expression of dipA or CTL target epi-position, because not derivative cell does not contain reproducible vaccinia virus, and reproducible vaccinia virus may discharge by non-specific side effect.The recombinant chou of selecting can further be expanded in compensation clone or under the situation that is having compensation helper virus or transfection plasmid to exist.

The early stage bovine vaccine gene of many keys is existing to be described.The preferred bovine vaccine strain of adopting the D4R genetic flaw.Bovine vaccine D4R open reading frame (orf) coding uracil dna glycosylase.This enzyme is that viral dna replication is necessary, and destroying this gene pairs bovine vaccine is lethal (A.K.Millns, M.S.Carpenter and A.M.Delange.1994 Virology 198:504-513).Someone is verified, and the stable transfection mammal cell line of expressing bovine vaccine D4R gene can compensate D4R defective type vaccinia virus (G.W.Holzer, F.G.Falkner.1997 J.Virology 71:4997-5002).In the absence of D4R compensation, infect with the bovine vaccine of D4R defective type and to cause that inhibition significantly reduces (Holzer and Falkner) to host cell proteins matter synthetic.Also show, the intragenic alien gene of tk that inserts the bovine vaccine of D4R defective type can continue by high level expression, even (M.Himly also like this when not having the D4R compensation, M.Pfleiderer, G.Holzer, U.Fischer, E.Hannak, F.G.Falkner and F.Dorner.1998 Protein Expression andPurification 14:317-326).Therefore, the D4R strain of replication defective is suitable for selecting those to rely on the active continuously virus recombinant of realizing its physiological action of expressing of some host gene very much.

To select special recombinant chou in the representative cDNA library of this strategy from be implemented in D4R defective type bovine vaccine strain in order utilizing, to need following clone and carrier:

1. the compensation clone that needs to express D4R is expanded D4R defective virus liquid storage.

2. the D4R gene that is applicable to the virus strain of three molecular recombination must deleted or deactivation.

3. must prepare the plasmid or the virus formulation body of expressing D4R under different inducible promoters regulation and control, described promotor comprises the promotor such as derivative other expression of gene of regulation and control BAX or the crosslinked back of the membrane immunoglobulin acceptor on CH33 B lymphoma cell; Perhaps after inducing the C3H10T1/2 progenitor cell to be divided into the chondrocyte, express the promotor of X collagen type.Need stable transfection of these constructs in relevant cell system to save special recombinant chou.Perhaps, can adopt the helper virus of expressing related constructs in clone or primary culture, to carry out abduction delivering.

10.1 the structure of D4R compensation clone.Following structure D4R compensation clone.At first, utilize following primer from bovine vaccine strain v7.5/tk, to clone D4R orf (100732 to 101388 of bovine vaccine genomes) through pcr amplification.

D4R-has justice, and 5 ' AAAGAATTCA TAATGAATTC AGTGACTGTA TCACACG3 ' is called SEQ ID NO:104 in the literary composition;

With the D4R-antisense, 5 ' CTTGGATCCT TAATAAATAA ACCCTTGAGC CC 3 ' is called SEQ ID NO:105 in the literary composition.

Have adopted primer to be modified to and comprise the EcoRI site, antisense primer is modified to and comprises BamHI site (all having added underscore represents).Carry out the standard pcr amplification also with after EcoRI and the BamHI digestion, the D4R orf that obtains is cloned into pIRESneo, and (available from Clontech, Palo Alto is CA) in the EcoRI in and the BamHI site.This mammalian expression vector contains strong CMV immediate early promoter/enhanser and ECMV internal ribosome entry site (IRES).The transfection of D4R/IRESneo construct in the BSC1 cell, is selected the transfection clone with G418.IRES can make 5 ' end contain the polycistronic mRNA that D4R orf, 3 ' end contain neomycin phosphotransferase and be translated efficiently.This makes that high-frequency G418 resistance clone all is (these clonal expressions D4R) that function is arranged.Detect transfection clone as probe through the Northern trace with the D4R gene, so that identify the clone of high level expression D4R mRNA.The BSC1 cell (BSC1.D4R) of expressing D4R can compensate the bovine vaccine of D4R defective type, makes to produce and to breed the D4R defective virus.

10.2.D4R the structure of defective type bovine vaccine carrier.Be suitable for as described in front embodiment 5, carrying out the D4R defective type vaccinia virus of three molecular recombination by following structure: interrupt D4R orf (genomic 100732 to 101388 of bovine vaccine) thereby intestinal bacteria GusA expression cassette is inserted the 300bp deletion fragment.

In order to insert the GusA gene, following side areas from vaccinia virus amplification insertion site.The following primer amplification of the flanking region on the left side:

There is justice in the D4R left side: 5 ' AATAAGCTTT ACTCCAGATA ATATGGA 3 '; Be called SEQ ID NO:106 in the literary composition;

D4R left side antisense: 5 ' AATCTGCAGC CCAGTTCCAT TTT 3 ' is called SEQ ID NO:107 in the literary composition.

These primer amplifications extend to a zone of 100960 from 100167 of bovine vaccine genomes, thereby they have been modified and have been comprised HindIII (justice is arranged) and PstI (antisense) site (all underlining expression) that is used to clone.Digest the PCR product that obtains with HindIII and PstI, be cloned into HindIII and the PstI site of pBS (available from Stratagene), produce the side areas of pBS.D4R.LF. with following primer amplification the right: there is justice on the D4R right side: 5 ' AATGGATCCT CATCCAGCGG CTA 3 ' is called SEQ ID NO:108 in the literary composition; D4R right side antisense: 5 ' AATGAGCTCT AGTACCTACA ACCCGAA 3 ' is called SEQ IDNO:109 in the literary composition.

These primer amplifications to one extend to 101975 zone from bovine vaccine genome 101271, thereby they have been modified and comprise BamHI (justice is arranged) and SacI (antisense) site (all the having underscore to represent) that is used to clone.With the PCR product that BamHI and SacI digestion obtain, be cloned into BamHI and the SacI site of pBS.D4R.LF, obtain pBS.D4R.LF/RF.

By the following method, will comprise the expression cassette insertion pBS.D4R.LF/RF that synthesizes the GusA coding region that (E/L) promotor operably connects together in morning/late period with poxvirus.Described E/L promotor-Gus box derives from pEL/tk-Gus and purchases and build body, at Merchlinsky, and M. etc., Virology 238:444-451 has description in (1997).Digest pEL/tk-Gus with NotI, fill and lead up with the KlenoW fragment then,, self connect generation pEL/tk Gus (NotI-) again to remove Gus ATG upstream from start codon next-door neighbour's NotI site.From pEL/tk-Gus (NotI-), separate the E/L-Gus expression cassette with following primer through Standard PC R:

EL-Gus has justice: 5 ' AAAGTCGACG GCCAAAAATT GAAATTTT 3 ', be called in the literary composition SEQ ID NO:110 and

The EL-Gus antisense: 5 ' AATGGATCCT CATTGTTTGC CTCCC 3 ' is called SEQ ID NO:111 in the literary composition.

EL-Gus has adopted primer to contain a SalI site, and the EL-Gus antisense primer contains a BamHI site (all being added with underscore).Behind the pcr amplification, the EL-Gus box digests with SalI and BamHI, inserts SalI and the BamHI site of pBS.D4R.LF/RF, produces pBS.D4R-/ELGus.This transferring plasmid contains the EL-Gus expression cassette that both sides are D4R sequences.Also in D4R orf, produced the disappearance of a 300bp.

After the transfection of pBS.D4R-/EL Gus construct is in the BSC1.D4R cell that has infected v7.5/tk-, be applicable to the D4R-/Gus+ vaccinia virus of three molecular recombination through conventional homologous recombination preparation.By on the BSC1.D4R cell, carry out plaque purification and with X-Glu dye separate D4R-/Gus+ virus (M.W.Carroll, B.Moss.1995.Biotechniques19:352-355).This novel strain called after v7.5/tk/Gus/D4R.

Make up representative bovine vaccine cDNA library according to the method that embodiment 5 describes by three molecular recombination from the DNA of v7.5/tk/Gus/D4R with purifying, but reaction is to carry out in BSC1.D4R compensation clone.

10.3. the host cell of the D4R that is subjected to the inducible promoter regulation and control is expressed in preparation.Express the host cell of D4R gene when being prepared as follows inducible promoter and being induced.Preparation can be expressed in the inducible promoter regulation and control plasmid construction body of bovine vaccine D4R gene down.The example of inducible promoter includes, but are not limited to membrane immunoglobulin on the CH33 cell promotor (being used for antibody selects) that raised when crosslinked, for example the BAX promotor of describing among the embodiment 2 and 3 takes place.The primer D4R that describes with top 10.1 parts has justice and D4R antisense through pcr amplification bovine vaccine D4Rorf.As required these PCR primers are modified, made them comprise required restriction endonuclease sites.Adopt the known method of those of ordinary skills that D4R orf is cloned in the suitable carrier for expression of eukaryon (make can select stable transformed cells) then, operably link together with required any promotor.

Then with this construct transfection in proper host cell, those cells that are used to select antibody of describing of embodiment 2 and 3 for example.The D4R gene that has operably connected the BAX promotor is stabilized the ground transfection in suitable clone, for example CH33 clone, CH31 clone or WEHI-231 clone.The host cell that obtains is basically according to utilizing the library that is structured among the v7.5/tk/Gus/D4R to be used to produce antibody as described in the embodiment 3.The crosslinked of the antigen induction of film expression immunoglobulin molecules causes the expression of D4R gene product in the crosslinked cell to be induced on the host cell surface.The expression of D4R has compensated the defective of v7.5/tk/Gus/D4R genome (being used for preparing therein the library), thereby produces infectious viral particle.

Embodiment 11

DNA synthetic reversible inhibitor is to the attenuation of host's inhibition of poxvirus mediation

As previously discussed, need attenuation or defective virus to reduce cytopathic effect sometimes.Cytopathic effect in the virus infection may disturb and utilize host cell death (for example crosslinked inductive apoptosis) to come selection and the evaluation that immunoglobulin molecules is carried out.This class effect can be with DNA synthetic reversible inhibitor such as hydroxyl urea (HU) (Pogo, B.G. and S.Dales Virology, 1971.43 (1): 144-51) weaken.HU suppresses cell and viral DNA synthetic (Hendricks, S.P. and C.K.Mathews J Biol Chem, 1998.273 (45): 29519-23) by the deoxynucleoside acid precursor of capturing in the replication complex.Viral dna replication is suppressed transcribes blocking-up viral RNA in late period, but allows transcribing and translating under the early stage bovine vaccine promoter regulation (Nagaya, A., B.G.Pogo and S.Dales Virology, 1970.40 (4): 1039-51).Therefore handle with DNA synthetic reversible inhibitor (such as HU) and can detect crosslinked effect.Suitably behind the incubation, can remove HU by the washing host cell and suppress, thereby the virus replication circulation is continued, and then be recovered to infectious recombinant chou (Pogo, B.G. and S.DalesVirology, 1971.43 (1): 144-51).

Result among Fig. 9 shows, in C3H10T 1/2 ancester cell of handling with BMP-2 (skeletal form generation albumen-2) X collagen type (it is the sign that the chondrocyte breaks up) synthetic induce by bovine vaccine infect block, but the HU mediation the viral DNA synthetic is suppressed can save this proteic synthesizing.When removing HU with fresh culture cleaning culture, viral DNA assembling synthetic and infectious particles is carried out very fastly, and washing back 2 hours is separable infectious viral particle.

With WR vaccinia virus infection C3H10T 1/2 cell of MOI=1, under the situation that is with or without 2mM HU, add the BMP-2 of substratum or 400ng/ml after 1 hour.37 ℃ cultivate 21 hours again after, clean with fresh culture and to remove HU.Make infectious cycle continue 2 hours, so that the assembling of initial viral dna replication and infectious particles.The 24th hour, from remain on 4 kinds of cells under the different culture condition, extract RNA.Carrying out Northern with X collagen type specific probe analyzes.Not derivative C3H10T1/2 cell has mesenchyme ancester cell phenotype, and does not express X collagen type (first hurdle is played on a left side).Adding BMP-2 to not infected C3H10T 1/2 cell normally can induce it to be divided into mature chondrocytes, and express X collagen type (relatively first and second hurdles are played on a left side), can not induce synthetic X collagen type (left side third column) and add BMP-2 for C3H10T 1/2 cell that infects bovine vaccine.Under the situation that has 2mM HU to exist, BMP-2 even in C3H10T 1/2 cell that has infected vaccinia virus, also can induce synthetic X collagen type (the 4th hurdle, the left side).

This strategy that weakens the pathological changes caused by virus effect can be used for other viruses, other cell types, and can be used to select the immunoglobulin molecules of cell death inducing when for example crosslinked.

Embodiment 12

The structure in multiple specific human Fab's fragment library

Be prepared as follows the complete people's of coding the segmental polynucleotide of diversified IgF ab library.These Fab fragments comprise the variable region of heavy chain that connects together with the first constant region structural domain (VH-CH1), and match with light chain immunoglobulin.Through pcr amplification people VH (variable region of heavy chain), the gene of VK (kappa light chain variable district) and VL (lambda light chain variable district).For each of this three variable region gene families, make up plasmid recombinant library and vaccinia virus library the two.The upstream of constant region sequence, the CH1 structural domain or the κ constant region of light chain CK of the corresponding heavy chain of described constant region sequence will be right after in the transfer/expression plasmid of variable region gene insertion based on p7.5/tk.Use these plasmids and produce corresponding vaccinia virus recombinant chou by three molecular recombination, also can be after the cell that has infected second immunoglobulin chain or its segmental vaccinia virus recombinant chou, with the direct high level expression Fab of these plasmids fragment with 1 immunoglobulin chain or its fragment transfection.These two chains are synthesized and assemble formation Fab fragment.Can understand as those of ordinary skills, by connecting the encoding sequence of signal sequence, membrane spaning domain and/or born of the same parents' intracellular domain, these Fab fragments can be film in conjunction with or secretor type.

12.1 pVHEc。Following structure coding comprises the expression vector of people's heavy chain fragment of the VH of C μ and CH1 structural domain, is called pVHEc.Description according to front embodiment 1.1 prepares plasmid p7.5/tk2.By DNA construct according to amino acid/11 09-113 and the CH1 structural domain (being the amino acid/11 09-223B of C μ) of embodiment 1 described isolating IgM heavy chain gene amplification coding VH, make it comprise a BstEII site through the PCR modification, comprise a terminator codon and a SalI site at 3 ' end at zone of coded amino acid 109-113+C μ CH1 structural domain 5 ' end.This DNA is inserted among the p7.5/tk2 between the BstEII and SalI, produce pVHEc.To utilize table 1 and 2 listed primers, be cloned into BssHII and BstEII site according to variable region of heavy chain (VH) the PCR product (amino acid (4) is to (110)) of the described preparation of embodiment 1.4 (a).Because it is overlapping that CH1 structural domain sequence and selected restriction enzyme sites exist, this will cause having made up the continuous heavy chain fragment that lacks functional signal peptide but be in the correct translation frame.

12.2 pVKEc and pVLEc.The expression vector of following structure coding people κ and lambda immunoglobulin constant region of light chain, called after pVKEc and pVLEc.As preparation plasmid p7.5/tk3.1 as described in the front embodiment 1.3.

(a) by the following method plasmid p7.5/tk3.1 is converted into pVKEc.The cDNA that separates coding C κ zone with primer according to the description of embodiment 1, make its 5 ' end comprise an XhoI site in the zone of coded amino acid 104-107+C κ, 3 ' end comprises a terminator codon and a SalI site, be cloned into XhoI and the SalI site of p7.5/tk3.1 then, produce pVKEc.Table 1 and 2 listed primers be will utilize then, ApaLI and the XhoI site of pVKEc will be cloned into according to kappa light chain variable district (VK) the PCR product (amino acid (3) is to (105)) of the described preparation of embodiment 1.4 (b).Owing to exist overlappingly between κ sequence of light chain and the selected restriction enzyme sites, this has caused making up a continuous κ light chain that lacks functional signal peptide but be in correct translation frame.

(b) by the following method plasmid p7.5/tk3.1 is converted into pVLEc.The cDNA that separates coding C κ zone with primer according to the description of embodiment 1, make its 5 ' end comprise a HindIII site in the amino acid/11 05-107 zone of coding V λ, comprise a terminator codon and a SalI site at 3 ' end, be cloned into HindIII and the SalI site of p7.5/tk3 then, produce pVLEc.Table 1 and 2 listed primers be will utilize then, ApaLI and the HindIII site of pVLEc will be cloned into according to lambda light chain variable district (VL) the PCR product (amino acid (3) is to (104)) of the described preparation of embodiment 1.4 (c).Owing to exist overlappingly between lambda light chain sequence and the selected restriction enzyme sites, this has caused making up a continuous lambda light chain that lacks functional signal peptide but be in correct translation frame.

12.3 the Fab of secretion or film combining form.Expression vector (pVHEc, pVKEc and pVLEc) can be used as the prototype carrier, and wherein the clone goes up secretion signal, membrane spaning domain, cytoplasmic structure territory or their combination and just Fab can be navigated to cell surface or born of the same parents' external space.The N-terminal that these signals and structural domain (table 7 has been listed their example) can insert Fab is in the SalI site of (perhaps between the NcoI and ApaLI of pVKEc and pVLEc) between the NcoI and BssHE of pVHEc and/or C-terminal.For the Fab target is secreted into the extracellular region chamber, at the Fab chain, one of VH-CH1 or light chain or both N-terminal are plugged a signal peptide.Carry out the outer submission of born of the same parents on the plasma membrane for Fab is anchored on, add a membrane spaning domain for the carboxyl terminal of VH-CH1 chain and/or light chain.Can also add the cytoplasmic structure territory.

Table 7. signal for locating
				Signal sequence	Terminal	The position	Protein
MGWSC IILFLVATATGAHS(S EQ ID NO：146)	N	ES	IgG1
				NLWTTASTFIVLFLLSLFYST TVTLF(SEQ ID NO：147)	C/N	PM	IgM

The abbreviation of the project under the position: ES, the outer pericentral siphon of born of the same parents; PM, plasma membrane.

Embodiment 13

The structure of people strand-Fv (ScFv) antibody library

13.1 as shown in figure 10, make up people scFv expression vector p7.5/tk3.2 and p7.5/tk3.3 by the following method.Description according to front embodiment 1.3 prepares plasmid p7.5/tk3.By changing 4 Nucleotide ATAC between NcoI and the ApaLI into ATAGC, p7.5/tk3 is converted into p7.5/tk3.1 with plasmid, and the initiator codon ATG among the NcoI does not conform to the ApaLI frame when having the signal peptide that inserts like this.Can realize easily transforming by the NotI-to-SaII box (SEQ ID NO:29) that apparatus has the box of sequence 5 '-GCGGCCGCCC ATGGATAGCG TGCACTTGAC TCGAGAAGCT TAGTAGTCGAC-3 ' (being called SEQ ID NO:112 in the literary composition) to replace describing among the embodiment 1.3.

By (being the Nucleotide 30 to 51 of SEQ ID NO:12 with following sequence box: XhoI-(Nucleotide of the amino acid/11 06-107 of coding V κ)-(Nucleotide of coding ten amino acid joint)-G-BssHII-ATGC-BstEII-zone that (Nucleotide of the amino acid/11 11-113 of coding VH)-terminator codon-SalI substitutes between XhoI and the SalI, be called SEQ ID NO:113 in the literary composition), p7.5/tk3.1 is converted into p7.5/tk3.2 with plasmid.This is by with XhoI and SalI digestion p7.5/tk3.1, and to insert a sequence be that the box (being called SEQ ID NO:114 in the literary composition) of 5 ' CTCGAGAT CAAAGAGGGT AAATCTTCCG GATCTGGTTC CGAAGGCGCGCATGCGGTCA CCGTCTCCTC ATGAGTCGAC 3 ' is realized.The final size of joint between V κ and the VH is 14 amino acid, and 4 wherein last amino acid are from the VH PCR product of following insertion.The sequence of joint is 5 ' GAGGGT AAA TCT TCC GGA TCT GGT TCC GAA GGC GCG CAC TCC, 3 ' (SEQID NO:115), its coded amino acid EGKSSGSGSEGAHS (SEQ ID NO:116).

By substituting the zone (being the Nucleotide 36 to 51 of SEQ ID NO:112) (being called SEQ ID NO:117 in the literary composition) between HindIII and the SalI with following sequence box: HindIII-(Nucleotide of the amino acid/11 05-107 of coding V λ)-(Nucleotide of coding ten amino acid joint)-G-BssHII-ATGC-BstEII-(Nucleotide of the amino acid/11 11-113 of coding VH)-terminator codon-SalI, p7.5/tk3.1 is converted into p7.5/tk3.3 with plasmid.This is by with HindIII and SalI digestion p7.5/tk3.1, and to insert a sequence be that the box (being called SEQ ID NO:118 in the literary composition) of 5 ' AAGCTTACCG TCCTAGAGGG TAAATCTTCC GGATCTGGTTCCGAAGGCGCG CATGCGGTCA CCGTCTCCTC ATGAGTCGAC 3 ' is realized.The final size of joint between V λ and the VH is 14 amino acid, and 4 wherein last amino acid are from the VH PCR product of following insertion.The sequence of joint is 5 ' GAG GGT AAA TCT TCC GGA TCT GGT TCC GAA GGC GCGCAC TCC, 3 ' (SEQ ID NO:119), its coded amino acid EGKSSGSGSEGAHS (SEQ ID NO:120).

13.2 the kytoplasm form of scFv.The scFv polypeptide expression carrier that following structure coding comprises people κ or lambda immunoglobulin variable region of light chain and merges with people's variable region of heavy chain.

(a) following structure kytoplasm V κ VH scFv expression product.With the primer that utilizes table 1 and 2 to list, be cloned between the ApaLI and XhoI site among the p7.5/tk3.2 according to kappa light chain variable district (V κ) the PCR product (amino acid (3) is to (105)) of the description of embodiment 1.4 (b) preparation.Because it is overlapping that κ sequence of light chain and selected restriction enzyme sites exist, this causes constructing and the identical continuous κ light chain of downstream joint translation frame.With the primer that utilizes table 1 and 2 to list, variable region of heavy chain (VH) PCR product (amino acid (4) is to (110)) according to the description of embodiment 1.4 (a) preparation is cloned between the BssHII and BstEII site of p7.5/tk3.2, forms a complete scFv open reading frame.The product that obtains is the V κ-VH fusion rotein by the kytoplasm form of 14 amino acid joints connection.The ScFv front is positioned at N-terminal 6 additional amino acid and part of V κ signal peptide by restriction site coding in addition.

(b) following structure kytoplasm V λ VH scFv expression product.With the primer that utilizes table 1 and 2 to list, be cloned between the ApaLI and HindIII site among the p7.5/tk3.3 according to lambda light chain variable district (VL) the PCR product (amino acid (3) is to (104)) of the description of embodiment 1.4 (c) preparation.Because it is overlapping that lambda light chain sequence and selected restriction enzyme sites exist, this causes constructing and the identical continuous lambda light chain of downstream joint translation frame.With the primer that utilizes table 1 and 2 to list, variable region of heavy chain (VH) PCR product (amino acid (4) is to (110)) according to the description of embodiment 1.4 (a) preparation is cloned between the BssHII and BstEII site of p7.5/tk3.3, forms a complete scFv open reading frame.The product that obtains is the V λ-VH fusion rotein by the kytoplasm form of 14 amino acid joints connection.The ScFv front is positioned at N-terminal 6 additional amino acid and part of V λ signal peptide by restriction site coding in addition.

13.3 the scFv of secretion or film combining form.13.2 the kytoplasm scFv expression vector that part is described can be used as the prototype carrier, just scFv can be navigated to cell surface or born of the same parents' external space after wherein being cloned into secretion signal, membrane spaning domain, cytoplasmic structure territory or their combination.Table 7 has been listed the example in signal peptide and film anchoring structure territory.For the scFv polypeptide is secreted into born of the same parents' external space, the box of an in-frame secreting signal peptide of coding is inserted between the NcoI and ApaLI site of p7.5/tk3.2 or p7.5/tk3.3, make it be expressed in the N-terminal of scFv polypeptide.Be used for film mating type scFv crosslinked based on Ig-or that the Ig-bonded is selected in order to produce, except signal peptide, the box of C μ of film combining form of also will encoding is cloned between the C-terminal BstEII and SalI site of scFv, be in the nucleotides downstream of coding VH amino acid/11 11-113 and with it frame conform to.Can also add a cytoplasmic structure territory.

Embodiment 14

The structure in people's single-chain antibody library of camelization

The Camelid species only form the antibody that is called as heavy chain antibody with heavy chain.The pox viruses express system can be used to produce secretor type or library, membrane-bound people's single structure territory, people V wherein _HStructural domain quilt " camelization " promptly is changed the V with camelid antibody _HThe H structural domain is identical, can/combination crosslinked according to Function detection or Ig-select then.Can be by conventional mutation method with people V _HThe gene camelization makes it more near camelidV _HThe H gene.For example, by replacing G44 with E, R replaces L45, and G or I replace W47, the suitable primer that utilization can be selected from table 1 and 2 to, according to the people V of the description preparation of embodiment 1.4 _H3 gene camelizations.Referring to for example Riechmann, L., and Muyldermans, S.J.Immunol.Meth.231:25-38.In order to produce secretor type single domain antibody library, with the people V of coding camelization _HThe box of gene is cloned into BssHII and the BstEII site of the pVHEs of preparation among the embodiment 1.2, and frame is expressed with conforming to.In order to produce membrane-bound single domain antibody library, with the people V of coding camelization _HThe box of gene is cloned into BssHII and the BstEIl site as the pVHE of preparation as described in the embodiment 1.1, and frame is expressed with conforming to.The existing signal peptide that is cloned between NcoI and BssHII site of carrier pVHE and pVHEs.People V to camelization _HThe amino-acid residue in three CDR districts carries out randomization widely in the gene, as described herein in poxvirus then the gained library is selected.

Embodiment 15

Select the adorned antibody of Fc of immune effector increased functionality

Human monoclonal antibodies just is being used to medical applications and is treating increasing human diseases.People's antibody may be by specific cell receptor-inducible or disabling signal conduction.In some applications, people's antibody may activate many secondary effects cells by the Fc part of antibody molecule and the interaction between the coupling Fc acceptor (FcR) on these effector cells.Therefore identify the combination that those can strengthen or suppress the FcR mediation and signal conducts or the modification of combination of other media (such as the composition of complement system) of immunological effect subfunction and activated immunoglobulin heavy chain constant region sequence is very interesting.Referring to for example United States Patent (USP) 5,624,821; Xu, D. etc., CellImmunol 200:16-26 (2000); With United States Patent (USP) 6,194,551, these documents are introduced herein as a reference in full.

Such specific response subfunction is antibody dependent cellular cytotoxicity (ADCC), and the target cell of coated antibody is destroyed by NK cell or other monocytes in this process.ADCC is mediated by such antibody molecule, and this molecule has coding has specific variable region and coding at the Fc γ RIII on the NK cell specific constant region to be arranged at the target cell surface molecular.By crystal structure analysis and rite-directed mutagenesis, determined that the Fc γ RIII binding site on the human IgG1 mainly is positioned at hinge down, i.e. about amino acid 247-252 of IgG1, and contiguous CH2 zone.Referring to for example Sarmay G. etc., Mol Immunol 29:633-639 (1992); And Michaelsen, T.E. etc., Mol Immunol 29:319-26 (1992).By in selectable mammalian expression vector, making up the gene library of antibody molecule that coding has the following hinge area of random mutation, just might choose special constant region variant with enhanced ADCC function.In order to simplify this implementation of strategies, make up a library with the specific definite immunoglobulin variable domain sequence of the purpose of giving.

15.1.pVHE-X and the structure of pVKE-X or pVLE-X.Following structure plasmid pVHE-X (have the people VH expression vector of definite variable region, be called X in the literary composition).Figure 11 has set forth building process.Separate by ordinary method, perhaps utilize poxvirus vector in eukaryotic cell, to prepare and select to have the antibody of specific specificity X by the method for describing in the literary composition.If necessary, between the VH gene subclone of the antibody BssHa/BstEII site in the pVHE of preparation as described in embodiment 1.1, produce plasmid pVHE-X.Equally if desired, the VK or the VL gene of antibody can be distinguished the ApaLI/XhoI site of subclone to the pVKE of preparation as described in embodiment 1.3, produce pVKE-X, perhaps subclone produces pVLE-X to the ApaLI/HindIII site of the pVLE of preparation as described in embodiment 1.3.

15.2 the separation of people C γ 1 box.Use following primer, use SMART ^TMThe RACEcDNA amplification kit separates the cDNA of coding people C γ 1 heavy chain from marrow RNA:

huCγ1-5B：5’ATTAGGATCC GGTCACCGTC TCCTCAGCC3’(SEQ IDNO：121)

huCγ1-3S：5’ATTAGTCGACTCATTTACCC GGAGACAGGG AGAG3’(SEQID NO：122).

The PCR product comprises following element: BamHI-BstEII-(Nucleotide of the amino acid/11 11-113 of coding VH)-(Nucleotide of the amino acid/11 14-478 of coding C γ 1)-TGA-SalI.With BamHI and the SalI site of this product subclone to pBluescriptII/KS, remove second BstEII site of amino acid/11 91 in corresponding the CH1 structural domain of C γ 1 and 192 by rite-directed mutagenesis, do not change aminoacid sequence.

15.3 the structure in Fc γ 1 library.Prepare C γ 1 variant through overlapping PCR by the following method.C γ 1 box that will suddenly change as the BstEII-of preparation as described in 15.2 parts is as the template of first round PCR, and amplification has justice/C γ 1-inside-R and C γ 1-inside-S/C γ 1-reverse primer group to carry out in two independent reactions with C γ 1-.

C γ 1-has justice: 5 ' AATATGGTCACCGTCTCCTCAGCC3 ' (SEQ ID NO:123)

C γ 1-inside-R:5 ' (MNN) ₆TTCAGGTGCTGGGCACGG3 ' (SEQ ID NO:124)

C γ 1-inside-S:5 ' (NNK) ₆GTCTTCCTCTTCCCCCCA3 ' (SEQ ID NO:125)

C γ 1-is reverse: 5 ' AATATGTCGACTCATTTACCCGG3 ' (SEQ ID NO:126)

(M＝A+C，K＝G+T，N＝A+T+G+C)

C γ 1-inside-R and C γ 1-inside-S primer have the degenerate sequence afterbody of the variant of coding six amino acid, and described six amino acid comprises the residue 247-252 of hinge down.Take turns among the PCR second, have justice and C γ 1 reverse primer the purified product of the first round to be merged through overlapping PCR with C γ 1.

The product volume that obtains is about 1000bp, at 20 all seed amino acids of each position hat of amino acid 247-252.With BstEII and SalI digestion PCR product, and be cloned into BstEII/SaII and digest among the good pVHE-X (as preparation as described in 15.1 parts), produce pVHE-X-γ 1 variant library.Description as embodiment 5 utilizes three molecular recombination that these variants are imported vaccinia virus then.Unite the recombinant chou vaccinia virus of carrying light chain, this Fc γ 1 library will can be used for selecting those to give VHE-X-γ 1 expressing antibodies with the active Fc variant of enhanced ADCC.

15.4 other application.Except producing the variant of amino acid 247-252, other residues also participate in combination with Fc γ RIII such as the amino acid 278-282 of IgG1 and amino acid 346-351.Identify demonstrate active Fc γ 1 variant that occurs in amino acid 247-252 of enhanced ADCC after, can adopt same strategy to identify that in other two zones those demonstrate other sudden changes of the collaborative ADCC of enhancing function.

Same principle/technology can be used for identifying some variants like this, and they give other immunoglobulin heavy chain constant region isotypes in conjunction with different Fc acceptors with the enhanced effector function.In the preferred embodiment, target recipient comprises Fc γ RI (CD64), Fc γ RII-A (CD32), Fc γ RII-B1, Fc γ RII-B2, Fc γ RIII (CD16) and Fc ε RI.In other preferred embodiments, can select to strengthen complement component and Fc zone combine or the Fc mediation with the combining of placental membrane so that through the variant of placental transport.

Embodiment 16

Make up the heavy chain fusion rotein and assisted to select to infect the cell of specific immune globulin gene recombinant chou vaccinia virus

16.1 the structure of CHl-Fas.Make up the expression vector of encoding fusion protein by the following method, this fusion rotein comprises people's heavy chain CH1 structural domain of C μ, and the Fas that merges with it stride film and death domain, called after CH1-Fas in the literary composition.Figure 13 (a) illustrates this fusion rotein.

With the plasmid pVHE of BstEII and SalI digestion as preparation as described in the embodiment 1.1, the little dna fragmentation of the about 1.4kb of gel-purified.With the template of this small segment as the PCR reaction, the primer is forward primer CH1 (F)-5 ' ACACGGTCAC CGTCTCCTCAGGGAGTGC 3 ' (SEQ ID NO:127) and reverse primer CH1 (R) 5 ' AGTTAGATCTGGATCCTGGA AGAGGCACGT T 3 ' (SEQ ID NO:128) then.PCR product to about 320 base pairs of obtaining carries out gel-purified.

With forward primer FAS (F) 5 ' AACGTGCCTC TTCCAGGATC CAGATCTAAC 3 ' (SEQ ID NO:129) and reverse primer FAS (R) 5 ' ACGCGTCGAC CTAGACCAAGCTTTGGATTT CAT 3 ' (SEQ ID NO:130) from plasmid pBS-AP014.2 amplifications comprise the dna fragmentation of striding film and death domain of Fas.The PCR product of about 504 base pairs that obtain is through gel-purified.

The fragment of 320 and 504 base pairs that will obtain then is incorporated in the PCR reaction, utilizes the fusion fragment of about 824 base pairs of forward primer CH1 (F) and reverse primer FAS (R) preparation.Digest this fragment with BstEII and SalI, the fragment of 810 base pairs that obtain is through gel-purified.Also with BstEII and SalI digestion, the big fragment of the about 5.7kb that obtains is through gel-purified for plasmid pVHE.Then these two BstEII/SalI fragments are connected and produce CH1-Fas.

16.2 the structure of CH4-Fas.Make up the expression vector of encoding fusion protein by the following method, this fusion rotein comprises people's heavy chain CH1-CH4 structural domain of C μ, and the Fas that merges with it stride film and death domain, called after CH4-Fas in the literary composition.Figure 13 (b) illustrates this fusion rotein.

With the plasmid pVHE of BstEII and SalI digestion as preparation as described in the embodiment 1.1, the little dna fragmentation of the about 1.4kb of gel-purified.With the template of this small segment as the PCR reaction, the primer is forward primer CH4 (F) 5 ' CTCTCCCGCG GACGTCTTCG T 3 ' (SEQ ID NO:131) and reverse primer CH4 (R) 5 ' AGTTAGATCT GGATCCCTCAAAGCCCTCCT C 3 ' (SEQ ID NO:132) then.The PCR product of about 268 base pairs that obtain is through gel-purified.

With the reverse primer FAS (R) in forward primer FAS (F2) 5 ' GAGGAGGGCT TTGAGGGATC CAGATCTAAC3 ' (SEQ ID NO:133) and 16.1 parts, comprise the dna fragmentation of striding film and death domain of Fas from plasmid pBS-AP014.2 amplification.The PCR product of about 504 base pairs that obtain is through gel-purified.

The fragment of 268 and 504 base pairs that will obtain then is incorporated in the PCR reaction, utilizes the fusion fragment of about 765 base pairs of forward primer CH4 (F) and reverse primer FAS (R) preparation.Digest this fragment with SacII and SalI, the fragment of 750 base pairs that obtain is through gel-purified.Also with SacII and SalI digestion, the big fragment of the about 6.8kb that obtains is through gel-purified for plasmid pVHE.Then these two SacII/SalI fragments are connected and produce CH4-Fas.

16.3 the structure of CH4 (TM)-Fas.Make up the expression vector of encoding fusion protein by the following method, this fusion rotein comprises people's heavy chain CH1-CH4 structural domain and the membrane spaning domain of C μ, and the death domain of the Fas that merges with it, called after CH4 (TM)-Fas in the literary composition.Figure 13 (c) illustrates this fusion rotein.

With the plasmid pVHE of BstEII and SalI digestion as preparation as described in the embodiment 1.1, the little dna fragmentation of the about 1.4kb of gel-purified.With the template of this small segment as the PCR reaction, the primer is the forward primer CH4 (F) and reverse primer CH4 (R2) 5 ' the AATAGTGGTG ATATATTTCA CCTTGAACAA 3 ' (SEQ ID NO:134) of 16.2 parts then.The PCR product of about 356 base pairs that obtain is through gel-purified.

The dna fragmentation that comprises the death domain of Fas with the reverse primer FAS (R) in forward primer FAS (F3) 5 ' TTGTTCAAGG TGAAAGTGAA GAGAAAGGAA3 ' (SEQ ID NO:135) and 16.1 parts from plasmid pBS-AP014.2 amplification.The PCR product of about 440 base pairs that obtain is through gel-purified.

The fragment of 356 and 440 base pairs that will obtain then is incorporated in the PCR reaction, utilizes the fusion fragment of about 795 base pairs of forward primer CH4 (F) and reverse primer FAS (R) preparation.Digest this fragment with SacII and SalI, the fragment of 780 base pairs that obtain is through gel-purified.Also with SacII and SalI digestion, the big fragment of the about 6.8kb that obtains is through gel-purified for plasmid pVHE.Then these two SacII/SalI fragments are connected, produce CH4 (TM)-Fas.

16.4 with various VH gene clones and insertion Ig-Fas fusion rotein.To utilize table 1 and 2 listed primers, be cloned into CH1-Fas, the BssHII of CH4-Fas and CH4 (TM)-Fas and BstEII site as variable region of heavy chain (VH) the PCR product (amino acid (4) is to (110)) of preparation as described in the embodiment 1.4 (a).Because it is overlapping that CH1 structural domain sequence and selected restriction enzyme sites exist, this will be built into the continuous heavy chain fragment that lacks functional signal peptide but be in the correct translation frame.

Embodiment 17

Express Ig α and the HeLaS3 of Ig β and the preparation of COS7 clone

For at the cell surface expression human monoclonal antibody specific, heavy chain and light chain immunoglobulin (Ig) must with other albumen physical connections of B-cell receptor complex body.Therefore, for make host cell can expressing human antibody library, they must be expressed and antibody synthesized effectively and be assembled into needed molecule of membrane-bound receptor and structure.Mouse lymphoma cell can be expressed in needed molecule of cell surface expression human antibodies specific and structure.But a shortcoming that lymphoma cell is used for the expression of people's antibody library is, the heavy chain immunoglobulin of endogenous expression and/or light chain can assemble altogether with the transgenosis immunoglobulin chain, cause forming nonspecific heterologous molecule, it will dilute antigen-specific receptor.It is very weak to come another shortcoming of expressing human antibody library to be that vaccinia virus is duplicated in lymphocyte series with mouse lymphoma cell.Therefore, the cell type that is preferred for expression specificity people antibody be those to allow to produce high titre vaccinia viruss and those be not cell derived from B clone.Preferred cell type comprises the HeLa cell, COS7 cell and BSC-1 cell.

The heavy chain immunoglobulin of B-cell receptor be in the same place with the heterodimer physical connection of Ig β transmembrane protein with light chain and Ig α (Reth, M.1992.Annu.Rev.Immunol.10:97).This physical connection is membrane bound immunoglobulin transporte to cells surface and carry out signal conduction necessary (Venkitaraman, A.R etc., 1991.Nature 352:777) through B-cell receptor effectively.But not clear Ig α/Ig β heterodimer is for expressing in allos clone whether film mating type immunoglobulin (Ig) is necessary and sufficient.Therefore, behind personnel selection Ig α and Ig β cDNA transfection HeLaS3 and the COS7 cell, the expression on these cell surfaces is assessed to people's antibody.

17.1 by PCR human cloning Ig α and Ig β cDNA.Will be from the cDNA of the human B cell preparation that transforms through EBV as the template of PCR reaction increase people Ig α and Ig β cDNA.With following primer amplification people Ig α cDNA:

Ig α 5 '-5 ' ATTAGAATTCATGCCTGGGGGTCCAGGA3 ' (SEQ ID NO:136); With

igα3’-5’ATTAGGATCCTCACGGCTTCTCCAGCTG3’(SEQ ID NO：137)。

With following primer amplification people Ig β cDNA:

igβ5’-5’ATTAGGATCCATGGCCAGGCTGGCGTTG3’(SEQ ID NO：138)；

igβ3’-5’ATTACCAGCACACTGGTCACTCCTGGCCTGGGTG3’(SEQ IDNO：139)。

With EcoRI and the BamHI site of Ig α PCR product cloning, and will arrive the BamHI and the BstXI site of pIREShyg carrier (Clontech) from the product cloning of Ig β PCR reaction to pIRESneo expression vector (Clontech).Confirm Ig α and the Ig β be cloned into by dna sequencing.

17.2 set up the HeLaS3 and COS7 stable transfection of expressing Ig α and Ig β.Utilize LIPOFECTAMINE PLUS Reagent (Life technologies), with the purifying pIRESneo-Ig α of 0.5 to 1 μ g and pIREShygIg β plasmid DNA transfection HeLaS3 and COS7 cell (in 6 orifice plates 1 * 10 ⁶/ hole).Begin after 2 days, select about 2 weeks with G418 (in 0.4mg/ml) and hygromycin B (in 0.2mg/ml).Directly separate resistance HeLaS3 bacterium colony, by limited dilution cloning COS7 transfectant.Analyze these clones' Ig α and Ig β expression then by RT-PCR, representative clone the results are shown in Figure 14.

Embodiment 18

Make up the diversified library of high affinity human antibody

The present invention is existing unique method that makes up immunoglobulin gene variation library in bovine vaccine or other poxvirus that is used in.Can design the bovine vaccine carrier and make its high level expression membrane receptor, thereby can be attached to effectively on the matrix of envelope antigen.Perhaps, can be with recombinant chou immunoglobulin heavy chain gene through engineering approaches, so that at acceptor cell death inducing during by antigen cross-linking.Even because vaccinia virus can be at an easy rate high efficiente callback in the cell that programmed cell death takes place, the unique features of this system makes it can select the specific human antibodies gene apace.

Best heavy chain immunoglobulin of select progressively and light chain can make the diversity maximization for heavy chain that utilizes and light chain combination by screening all.Sequentially screen strategy and at first containing 10 ⁴Under the situation that the little library of individual different light chains exists, from containing 10 ⁵Select best heavy chain in the little library of individual H chain recombinant chou.Bigger contain 10 with the H chain of this optimization from one then ⁶To 10 ⁷Select its best partner in the library of individual recombinant chou L chain.In case choose best L chain, just can contain 10 from bigger ⁶To 10 ⁷Select better H chain in the individual recombinant chou H chain library.This is a kind of shoestring strategy repeatedly, and it can be from reaching 10 ¹⁴Plant H ₂L ₂Choose specific high-affinity antibody in the combination.On the contrary, selecting strand Fv or be chosen in the Fab that is made of independent VH-CH1 and VL-CL gene that encodes on the single plasmid in phage library is a one-step process, and perhaps it is subjected to single phage library actual size (is 10 ¹¹Individual phage particle) restriction.

Because screen 10 ⁷Plant H chain and 10 ⁷Plant 10 of L chain ¹⁴Kind the combination be infeasible, so for the selection best H chain, during beginning in noninfectious vector is arranged 10 ⁴Plant under the situation of L chain existence, from 10 ⁵Select in the library of H chain bovine vaccine recombinant chou.These combinations produce the low-affinity antibody of many epi-positions probably, cause choosing for example 1 to 100 different H chain.If choose 100 H chains for a basic antibody, they can be used for 10 then ⁶Or 10 ⁷Carry out second in the bigger library of bovine vaccine recombinant chou L chain and take turns selection, 100 best L chain partners of picking.Shelve original H chain then, with these 100 L chains from 10 ⁶Or 10 ⁷Select the H chain of new more high-affinity in the bigger library of H chain.This strategy resembles a kind of external avidity increaseization.As the same in normal immunne response, at first select low-affinity antibody then in immunity circulation repeatedly conduct select more high-affinity offspring's basis.High-affinity clone may derive from intravital somatic mutation, and the recombine of this external strategy by immunoglobulin chain can reach same purpose.In the both of these case, the partner of the partner of the immunoglobulin chain of improvement and the low-affinity antibody of beginning is the same.

The basis of this strategy is control selection to low-affinity antibody when initial.It is very crucial to choose low-affinity antibody.The method based on bovine vaccine that is used for select progressively H and L chain can suitably be optimized, and it is successful selecting with the low-affinity of guaranteeing to begin, because it possesses the avidity advantage that bivalent antibody brings of expressing.In addition, by in the bovine vaccine system, adopting different promoters can regulate and control the level of antibody expression.For example, the T7 polysaccharase system of adaptation bovine vaccine produces higher levels of expression than natural bovine vaccine promotor.Begin several " basic antibody " that selection can guarantee to choose low-affinity based on high-level T7 expression system of taking turns, the back is several takes turns selection and can guide the derivative of selecting high-affinity based on low expression level.

Below be the summary that the present invention is used for making up in bovine vaccine the method in immunoglobulin gene variation library:

1. according to the method for describing in the literary composition, in vaccinia virus vector, make up immune globulin tunica albuginea dependency heavy chain cDNA library from human lymphocyte.Infect the cell of special through engineering approaches with the viral library (on average each cell is infected by a virus immunity sphaeroprotein heavy chain recombinant chou) of dilution, CH33 cell for example, murine myeloma cell and people EBV transformation cell lines, perhaps preferred HeLa cell and other not competing property immunoglobulin chain also can effectively be supported the non-lymphocyte that bovine vaccine is duplicated.

With the psoralene deactivation from same these cells of the light chain immunoglobulin recombinant chou vaccinia virus infection in the light chain immunoglobulin library that is structured in the identical vaccinia virus vector.Perhaps, can be with these cells of light chain immunoglobulin recombinant chou transfection in the plasmid expression vector.In whole cell mass, each heavy chain can be associated with any light chain.

3. the time that cell cultures is suitable is so that the optimal expression of the antibody that obtains being fully assembled at cell surface.When host cell is not the lymph source, has adopted stable transfection and expressed the host cell of Ig α and proteic gene of Ig β or cDNA, for example Hela or Cos7 cell can improve the efficient that membrane antibody is expressed.

4a. purpose antigen is attached on the inertia pearl, then the latter is mixed with antibody expression cell library.Recovery is attached to the cell on the pearl of envelope antigen, extracts relevant heavy chain immunoglobulin recombinant virus.

4b. or, connect fluorescence labels directly or indirectly for purpose antigen.Reclaim and antigen bonded antibody expression cell by fluorescence-activated cell sorting.

4c. or, can adopt those antibody receptors and antigenic crosslinked host cell with inducing cell death.Spontaneous generation when this may be the immature cell of host cell B cell kind system, or introduce the result of the death domain of Fas coding at the carboxyl terminal of immunoglobulin heavy chain constant region.Lysing cell and viable cell are separated, extract the recombinant virus that carries corresponding heavy chain immunoglobulin.

5. the circulation with above step 1-4 repeats several times, separates recombinant virus at every turn and further be enriched with to be beneficial to best antigen bonded heavy chain.

In a single day 6. choose the specific antibody heavy chain, to being structured in the light chain immunoglobulin cDNA library whole process repeated in the monopoly vaccinia virus, so that select to help best antigen bonded specific immune sphaeroprotein light chain.Can make up select progressively heavy chain and light chain for the heavy chain and the light chain that utilize by screening all, can reach variation to greatest extent.Bivalent antibody by selecting assembling fully but not strand Fv or monomer Fab optimize last Mab product.

7. determine the sequence of Mab and verify its specific combination through the standard test technology.

Bivalent antibody by selecting assembling fully but not strand Fv optimizes last Mab product.In other words, select to be based on divalence (H ₂L ₂) antibody but not scFv or Fab fragment.Synthetic and assemble complete people's complete antibody in mammalian cell, can make immunoglobulin chain can carry out normal posttranslational modification and assembling.Synthetic and the assembling of complete antibody may be very low in the bacterial cell internal efficiency, and can lose many specificitys, because a lot of antibody can not correctly fold in the abnormal physiology environment of bacterial cell.

Can choose antibody epitope specificity, comprise according to functionally active and select specificity than relative broad range.Specifically, can be according to the specific physiological effect of target cell being selected antibody (for example screen TNF excretory inhibition to activated monocyte; Cell death inducing etc.).The following Function detection that is based on is screened the method for special Mab and is summarized:

1. make up the not heavy chain immunoglobulin cDNA library of immune human lymphocyte in the vaccinia virus vector of the method preparation of in according to literary composition, describing.With for example about 100 increasing respectively to a plurality of ponds of about 1000 recombinant virus, be used for infecting producer's cell, its extent of dilution makes average each cell be infected by a heavy chain immunoglobulin recombinant virus.With the psoralene deactivation from same these cells of the light chain immunoglobulin recombinant chou vaccinia virus infection in the light chain immunoglobulin library that is structured in the identical vaccinia virus vector.Perhaps, can be with the infected cell of light chain immunoglobulin recombinant chou transfection in the plasmid expression vector.In whole cell mass, each heavy chain can be associated with any light chain.

2. with infected cell cultures time enough, make that the antibody of assembling is secreted fully.

3. set up and detect the hole, wherein incubation purpose function indicator cells under the situation that has the secretory antibody sample to exist.Described cell may for example comprise the activated monocyte of TNF secretion α.Can adopt simple T NF α ELISA to detect then and screen and comprise the active antibody of factor excretory capable of inhibiting cell pond.

4. the single member who further analyzes institute's scavenger identifies related immune sphaeroprotein heavy chain.

In a single day 5. choose the specific antibody heavy chain, to being structured in the light chain immunoglobulin cDNA library whole process repeated in the monopoly vaccinia virus vector, so that select to help best antigen bonded specific immune sphaeroprotein light chain.

6. come identification of M ab sequence and verify its specific combination by the standard test technology.Because functionally selected the needs knows about earlier the target film acceptor, also be the discovering tool of identifying the related film acceptor so the Mab that chooses is the potential medicine.

After heavy chain immunoglobulin and the light chain random incorporation, in people's cell culture, select.As above-mentioned, this has been avoided the limitation of the antibody group that the restriction of synthetic aspect in the bacterium causes.It can also avoid in mouse the restriction of the antagonist group that the tolerance to the homologous gene product causes.The mouse homologue of important human protein usually has 80% identical to 85% with the human sequence.Therefore, estimate that mouse antibodies reaction to human protein mainly concentrates on 15% to 20% different epi-position in people and the mouse.The invention enables the high-affinity that effectively to select to have the wide region epitope specificity, complete people's antibody.This technology can be used for multiple project and purpose, comprises that the antibody of not identifying membrane receptor to having specific physiological significance carries out functionally selected.

Scope of the present invention is not subjected to the restriction of described specific embodiments, and these embodiments are used for an illustration as indivedual aspects of invention, the construct that all functions are equal to, and virus and enzyme are all within the scope of the invention.Really, except showing in the literary composition and describe that according to the specification sheets and the accompanying drawing of front, various changes of the present invention are conspicuous to those of ordinary skills.These changes all fall within the scope of the appended claims.

All publications mentioned in the specification sheets and patent application all are incorporated herein by reference separately and particularly in full.Ask 60/192586 to introduce as a reference in the United States Patent (USP) that the U.S. Patent application 08/935377 that on September 22nd, 1997 submitted to and on March 28th, 2000 submit to herein.

Sequence table

<110>University of Rochester

＜120〉method of screening coding for antigens specific immunoglobulin molecule or the segmental polynucleotide of its antigen-specific

<130>1821.007PC05

<150>60/271,424

<151>2001-02-27

<150>60/262,067

<151>2001-01-18

<150>60/298,087

<151>2001-06-15

<150>60/249,268

<151>2000-11-17

<160>147

<170>PatentIn version 3.1

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＜213〉artificial sequence

<220>

＜223〉the unique BssHII site among the pVHE

<400>15

gcgcgc 6

<210>16

<211>7

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the unique BstEII site among the pVHE

<400>16

ggtcacc 7

<210>17

<211>446

<212>DNA

＜213〉artificial sequence

<220>

＜223〉pVKE transferring plasmid

<400>17

ggccaaaaat tgaaaaacta gatctattta ttgcacgcgg ccgcccatgg gatggagctg 60

tatcatcctc ttcttggtag caacagctac aggcgtgcac ttgactcgag atcaaacgaa 120

ctgtggctgc accatctgtc ttcatcttcc cgccatctga tgagcagttg aaatctggaa 180

ctgcctctgt tgtgtgcctg ctgaataact tctatcccag agaggccaaa gtacagtgga 240

aggtggataa cgccctccaa tcgggtaact cccaggagag tgtcacagag caggacagca 300

aggacagcac ctacagcctc agcagcaccc tgacgctgag caaagcagac tacgagaaac 360

acaaagtcta cgcctgcgaa gtcacccatc agggcctgag ctcgcccgtc acaaagagct 420

tcaacagggg agagtgttag gtcgac 446

<210>18

<211>6

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the unique ApaLI site in the pVKE plasmid

<400>18

gtgcac 6

<210>19

<211>6

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the unique XhoI site in the pVKE plasmid

<400>19

ctcgag 6

<210>20

<211>455

<212>DNA

＜213〉artificial sequence

<220>

＜223〉pVLE transferring plasmid

<400>20

ggccaaaaat tgaaaaacta gatctattta ttgcacgcgg ccgcccatgg gatggagctg 60

tatcatcctc ttcttggtag caacagctac aggcgtgcac ttgactcgag aagcttaccg 120

tcctacgaac tgtggctgca ccatctgtct tcatcttccc gccatctgat gagcagttga 180

aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga gaggccaaag 240

tacagtggaa ggtggataac gccctccaat cgggtaactc ccaggagagt gtcacagagc 300

aggacagcaa ggacagcacc tacagcctca gcagcaccct gacgctgagc aaagcagact 360

acgagaaaca caaagtctac gcctgcgaag tcacccatca gggcctgagc tcgcccgtca 420

caaagagctt caacagggga gagtgttagg tcgac 455

<210>21

<211>6

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the unique ApaLI site in the pVLE plasmid

<400>21

gtgcac 6

<210>22

<211>6

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the unique HindIII site among the pVLE

<400>22

aagctt 6

<210>23

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉H-2Kd restriction peptide (restricted peptide)

<400>23

Gly Tyr Lys Ala Gly Met Ile His Ile

1 5

<210>24

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>24

attaggatcc ggtcaccgtc tcctcaggg 29

<210>25

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>25

attagtcgac tcatttcacc ttgaacaagg tgac 34

<210>26

<211>47

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used to prepare the sequence box of p7.5/tk2

<400>26

gcggccgcaa accatggaaa gcgcgcatat ggtcaccaaa agtcgac 47

<210>27

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>27

attaggatcc ggtcaccgtc tcctcaggg 29

<210>28

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>28

attagtcgac tcagtagcag gtgccagctg t 31

<210>29

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used to prepare the sequence box of p7.5/tk3

<400>29

gcggccgccc atggatacgt gcacttgact cgagaagctt agtagtcgac 50

<210>30

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>30

caggactcga gatcaaacga actgtggctg 30

<210>31

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>31

aatatgtcga cctaacactc tcccctgttg aagctcttt 39

<210>32

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>32

aatatgtcga cctaacactc tcccctgttg aagctctt 38

<210>33

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>33

atttaagctt accgtcctac gaactgtggc tgcaccatct 40

<210>34

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>34

ttttgcgcgc actcccaggt gcagctggtg cagtctgg 38

<210>35

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>35

ttttgcgcgc actccgaggt gcagctggtg gagtctgg 38

<210>36

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>36

ttttgcgcgc actcccaggt gcagctgcag gagtcggg 38

<210>37

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>37

gacggtgacc agggtgccct ggcccca 27

<210>38

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>38

gacggtgacc agggtgccac ggcccca 27

<210>39

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>39

gacggtgacc attgtccctt ggcccca 27

<210>40

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>40

gacggtgacc agggttccct ggcccca 27

<210>41

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>41

gacggtgacc gtggtccctt ggcccca 27

<210>42

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>42

tttgtgcact ccgacatcca gatgacccagtctcc 35

<210>43

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>43

tttgtgcact ccgatgttgt gatgactcag tctcc 35

<210>44

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>44

tttgtgcact ccgaaattgt gttgacgcag tctcc 35

<210>45

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>45

tttgtgcact ccgacatcgt gatgacccag tctcc 35

<210>46

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>46

tttgtgcact ccgaaacgac actcacgcag tctcc 35

<210>47

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>47

tttgtgcact ccgaaattgt gctgactcag tctcc 35

<210>48

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>48

gatctcgagcttggtccctt ggccgaa 27

<210>49

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>49

gatctcgagc ttggtcccct ggccaaa 27

<210>50

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>50

gatctcgagt ttggtcccag ggccgaa 27

<210>51

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>51

gatctcgagc ttggtccctc cgccgaa 27

<210>52

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>52

aatctcgagt cgtgtccctt ggccgaa 27

<210>53

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>53

tttgtgcact cccagtctgt gttgacgcag ccgcc 35

<210>54

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>54

tttgtgcact cccagtctgc cctgactcag cctgc 35

<210>55

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>55

tttgtgcact cctcctatgt gctgactcag ccacc 35

<210>56

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>56

tttgtgcact cctcttctga gctgactcag gaccc 35

<210>57

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>57

tttgtgcact cccacgttatactgactcaa ccgcc 35

<210>58

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>58

tttgtgcact cccaggctgt gctcactcag ccgtc 35

<210>59

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>59

tttgtgcact ccaattttat gctgactcag cccca 35

<210>60

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>60

tttgtgcact cccaggctgt ggtgactcag gagcc 35

<210>61

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>61

ggtaagcttg gtcccagttc cgaagac 27

<210>62

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>62

ggtaagcttg gtccctccgc cgaat 25

<210>63

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>63

aatatgcgcg cactcccagg tgcagctggt gcagtctgg 39

<210>64

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>64

aatatgcgcg cactcccagg tcaccttgaa ggagtctgg 39

<210>65

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>65

aatatgcgcg cactccgagg tgcagctggt ggagtctgg 39

<210>66

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>66

aatatgcgcg cactcccagg tgcagctgca ggagtcggg 39

<210>67

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>67

aatatgcgcg cactccgagg tgcagctggt gcagtctg 38

<210>68

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>68

gagacggtga ccagggtgcc ctggcccca 29

<210>69

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>69

gagacggtga ccagggtgcc acggcccca 29

<210>70

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>70

gagacggtga ccattgtccc ttggcccca 29

<210>71

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>71

gagacggtga ccagggttcc ctggcccca 29

<210>72

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>72

gagacggtga ccgtggtccc ttggcccca 29

<210>73

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>73

caggagtgca ctccgacatc cagatgaccc agtctcc 37

<210>74

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>74

caggagtgca ctccgatgtt gtgatgactc agtctcc 37

<210>75

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>75

caggagtgca ctccgaaatt gtgttgacgc agtctcc 37

<210>76

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>76

caggagtgca ctccgacatc gtgatgaccc agtctcc 37

<210>77

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>77

caggagtgca ctccgaaacg acactcacgc agtctcc 37

<210>78

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>78

caggagtgca ctccgaaatt gtgctgactc agtctcc 37

<210>79

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>79

ttgatctcga gcttggtccc ttggccgaa 29

<210>80

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>80

ttgatctcga gcttggtccc ctggccaaa 29

<210>81

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>81

ttgatctcga gtttggtccc agggccgaa 29

<210>82

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>82

ttgatctcga gcttggtccc tccgccgaa 29

<210>83

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>83

ttaatctcga gtcgtgtccc ttggccgaa 29

<210>84

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>84

cagatgtgca ctcccagtct gtgttgacgc agccgcc 37

<210>85

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>85

cagatgtgca ctcccagtct gccctgactc agcctgc 37

<210>86

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>86

cagatgtgca ctcctcctat gtgctgactc agccacc 37

<210>87

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>87

cagatgtgca ctcctcttct gagctgactc aggaccc 37

<210>88

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>88

cagatgtgca ctcccacgtt atactgactc aaccgcc 37

<210>89

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>89

cagatgtgca ctcccaggct gtgctcactc agccgtc 37

<210>90

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>90

cagatgtgca ctccaatttt atgctgactc agcccca 37

<210>91

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>91

cagatgtgca ctcccaggct gtggtgactc aggagcc 37

<210>92

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>92

acggtaagct tggtcccagt tccgaagac 29

<210>93

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>93

acggtaagct tggtccctcc gccgaatac 29

<210>94

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>94

atgttacgtc ctgtagaaac c 21

<210>95

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>95

tcattgtttg cctccctgct g 21

<210>96

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>96

aaagcggccg ccccgggatg ttacgtcc 28

<210>97

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>97

aaagggcccg gcgcgcctca ttgtttgcc 29

<210>98

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>98

aaaggatcca taatgaattc agtgactgta tcacacg 37

<210>99

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>99

cttgcggccg cttaataaat aaacccttga gccc 34

<210>100

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>100

attgagctct taatactttt gtcgggtaac agag 34

<210>101

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>101

ttactcgaga gtgtcgcaat ttggatttt 29

<210>102

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>102

aaagaattcc tttattgtcatcggccaaa 29

<210>103

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>103

aatctgcagt cattgtttgc ctccctgctg 30

<210>104

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>104

aaagaattca taatgaattc agtgactgta tcacacg 37

<210>105

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>105

cttggatcct taataaataa acccttgagc cc 32

<210>106

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>106

aataagcttt actccagata atatgga 27

<210>107

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>107

aatctgcagc ccagttccat ttt 23

<210>108

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>108

aatggatcct catccagcgg cra 23

<210>109

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>109

aatgagctct agtacctaca acccgaa 27

<210>110

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>110

aaagtcgacg gccaaaaatt gaaatttt 28

<210>111

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>111

aatggatcct cattgtttgc ctccc 25

<210>112

<211>51

<212>DNA

＜213〉artificial sequence

<220>

＜223〉plasmid p7.5/tk3 is converted into the sequence box of p7.5/tk3.1

<400>112

gcggccgccc atggatagcg tgcacttgac tcgagaagct tagtagtcga c 51

<210>113

<211>22

<212>DNA

＜213〉artificial sequence

<220>

Substituted zone when＜223〉plasmid p7.5/tk3.1 being converted into p7.5/tk3.2

<400>113

ctcgagaagc ttagtagtcg ac 22

<210>114

<211>78

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used for plasmid p7.5/tk3.1 is converted into the sequence box of p7.5/tk3.2

<400>114

ctcgagatca aagagggtaa atcttccgga tctggttccg aaggcgcgca tgcggtcacc 60

gtctcctcat gagtcgac 78

<210>115

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉p7.5/tk3.2 joint

<400>115

gagggtaaat cttccggatc tggttccgaa ggcgcgcact cc 42

<210>116

<211>14

<212>PRT

＜213〉artificial sequence

<220>

＜223〉p7.5/tk3.2 joint

<400>116

Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Gly Ala His Ser

1 5 10

<210>117

<211>16

<212>DNA

＜213〉artificial sequence

<220>

Substituted zone when＜223〉plasmid p7.5/tk3.1 is converted into p7.5/tk3.3

<400>117

aagcttagta gtcgac 16

<210>118

<211>81

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used for plasmid p7.5/tk3.1 is converted into the sequence box of p7.5/tk3.3

<400>118

aagcttaccg tcctagaggg taaatcttcc ggatctggtt ccgaaggcgc gcatgcggtc 60

accgtctcct catgagtcga c 81

<210>119

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉p7.5/tk3.3 joint

<400>119

gagggtaaat cttccggatc tggttccgaa ggcgcgcact cc 42

<210>120

<211>14

<212>PRT

＜213〉artificial sequence

<220>

＜223〉p7.5/tk3.3 joint

<400>120

Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Gly Ala His Ser

1 5 10

<210>121

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>121

attaggatcc ggtcaccgtctcctcagcc 29

<210>122

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>122

attagtcgac tcatttaccc ggagacaggg agag 34

<210>123

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>123

aatatggtca ccgtctcctc agcc 24

<210>124

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<220>

＜221〉other characteristics

<222>(2)..(3)

＜223〉can be any Nucleotide

<220>

＜221〉other characteristics

<222>(5)..( 6)

＜223〉can be any Nucleotide

<220>

＜221〉other characteristics

<222>(8)..(9)

＜223〉can be any Nucleotide

<220>

＜221〉other characteristics

<222>(11)..(12)

＜223〉can be any Nucleotide

<220>

＜221〉other characteristics

<222>(14)..(15)

＜223〉can be any Nucleotide

<220>

＜221〉other characteristics

<222>(17)..(18)

＜223〉can be any Nucleotide

<400>124

mnnmnnmnnm nnmnnmnntt caggtgctgg gcacgg 36

<210>125

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<220>

＜221〉other characteristics

<222>(1)..(2)

＜223〉can be any Nucleotide

<220>

＜221〉other characteristics

<222>(4)..(5)

＜223〉can be any Nucleotide

<220>

＜221〉other characteristics

<222>(7)..(8)

＜223〉can be any Nucleotide

<220>

＜221〉other characteristics

<222>(10)..(11)

＜223〉can be any Nucleotide

<220>

＜221〉other characteristics

<222>(13)..(14)

＜223〉can be any Nucleotide

<220>

＜221〉other characteristics

<222>(16)..(17)

＜223〉can be any Nucleotide

<400>125

nnknnknnkn nknnknnkgt cttcctcttc ccccca 36

<210>126

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>126

aatatgtcga ctcatttacc cgg 23

<210>127

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>127

acacggtcac cgtctcctca gggagtgc 28

<210>128

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>128

agttagatct ggatcctgga agaggcacgt t 31

<210>129

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>129

aacgtgcctc ttccaggatc cagatctaac 30

<210>130

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>130

acgcgtcgac ctagaccaag ctttggattt cat 33

<210>131

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>131

ctctcccgcg gacgtcttcg t 21

<210>132

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>132

agttagatct ggatccctca aagccctcct c 31

<210>133

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>133

gaggagggct ttgagggatc cagatctaac 30

<210>134

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>134

aatagtggtg atatatttca ccttgaacaa 30

<210>135

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>135

ttgttcaagg tgaaagtgaa gagaaaggaa 30

<210>136

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>136

attagaattc atgcctgggg gtccagga 28

<210>137

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>137

attaggatcc tcacggcttc tccagctg 28

<210>138

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>138

attaggatcc atggccaggc tggcgttg 28

<210>139

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>139

attaccagca cactggtcac tcctggcctg ggtg 34

<210>140

<211>69

<212>DNA

＜213〉artificial sequence

<220>

＜223〉p7.5/tk promotor

<220>

<221>CDS

<222>(46)..(69)

<223>

<400>140

ggccaaaaat tgaaaaacta gatctattta ttgcacgcgg ccgcc atg ggc ccg gcc 57

Met Gly Pro Ala

1

gcc aac ggc gga 69

Ala Asn Gly Gly

5

<210>141

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the tk sequence of p7.5/tk

<400>141

Met Gly Pro Ala Ala Asn Gly Gly

1 5

<210>142

<211>75

<212>DNA

＜213〉artificial sequence

<220>

＜223〉pE/Ltk promotor

<220>

<221>CDS

<222>(52)..(75)

<223>

<400>142

ggccaaaaat tgaaatttta tttttttttt ttggaatata aagcggccgc c atg ggc 57

Met Gly

1

ccg gcc gcc aac ggc gga 75

Pro Ala Ala Asn Gly Gly

5

<210>143

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the tk sequence of pE/Ltk

<400>143

Met Gly Pro Ala Ala Asn Gly Gly

1 5

<210>144

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>144

aatatgcgcg cactcccagg tcaccttgaa ggagtctgg 39

<210>145

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>145

aatatgcgcg cactcccgagg tgcagctggt gcagtctg 38

<210>146

<211>19

<212>PRT

＜213〉artificial sequence

<220>

＜223〉signal sequence

<400>146

Met Gly Trp Ser cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Ala His Ser

<210>147

<211>26

<212>PRT

＜213〉artificial sequence

<220>

＜223〉signal sequence

<400>147

Asn Leu Trp Thr Thr Ala Ser Thr phe Ile Val Leu Phe Leu Leu Ser

1 5 10 15

Leu Phe Tyr Ser Thr Thr Val Thr Leu Phe

20 25

Claims (88)

1. method of selecting coding for antigens specific immunoglobulin molecule or the segmental polynucleotide of its antigen-specific comprises:

(a) will import to the eukaryotic host cell group that can express described immunoglobulin molecules through the first polynucleotide library that has operably connected transcription regulatory region, the multiple first immunoglobulin (Ig) subunit polypeptide of coding; Wherein each first immunoglobulin (Ig) subunit polypeptide comprises:

(i) be selected from first constant region for immunoglobulin of CH and constant region of light chain,

(ii) with the corresponding immune globulin variable region of described first constant region, and

(iii) can instruct the cell surface expression or the excretory signal peptide of the described first immunoglobulin (Ig) subunit polypeptide;

(b) in described host cell, import the second polynucleotide library of the multiple second immunoglobulin (Ig) subunit polypeptide of coding operably connected transcription regulatory region; Wherein each second immunoglobulin (Ig) subunit polypeptide comprises:

(i) be selected from second constant region for immunoglobulin of CH and constant region of light chain, wherein this second constant region for immunoglobulin is different with first constant region for immunoglobulin,

(ii) with the corresponding immune globulin variable region of described second constant region, and

(iii) can instruct the cell surface expression or the excretory signal peptide of the described second immunoglobulin (Ig) subunit polypeptide;

The wherein said second immunoglobulin (Ig) subunit polypeptide can be combined to form immunoglobulin molecules or its antigen-specific fragment with the described first immunoglobulin (Ig) subunit polypeptide;

(c) allow described host cell expression immunoglobulin molecules or its antigen-specific fragment;

(d) described immunoglobulin molecules is contacted with antigen; And

(e) those polynucleotide of the coding first immunoglobulin (Ig) subunit polypeptide in the recovery first polynucleotide library, the described first immunoglobulin (Ig) subunit polypeptide is special as an immunoglobulin molecules or the segmental part of its antigen-specific for described antigen.

2. the method for claim 1 comprises in addition:

(f) the described polynucleotide that are recovered to are imported the host cell group that can express described immunoglobulin molecules;

(g) import the described second polynucleotide library to this host cell;

(h) allow described host cell expression immunoglobulin molecules or its antigen-specific fragment;

(i) described host cell is contacted with antigen; And

(j) those polynucleotide of the coding first immunoglobulin (Ig) subunit polypeptide in the recovery first polynucleotide library, the described first immunoglobulin (Ig) subunit polypeptide is special as an immunoglobulin molecules or the segmental part of its antigen-specific for described antigen.

3. the method for claim 2, comprise in addition step (f)-(j) is repeated one or repeatedly, thereby the polynucleotide of the coding first immunoglobulin (Ig) subunit polypeptide in the described first polynucleotide library of enrichment, wherein this first immunoglobulin (Ig) subunit polypeptide can be specifically in conjunction with described antigen as an immunoglobulin molecules or the segmental part of its antigen-specific.

4. the method for claim 1 comprises those polynucleotide that separation is recovered in addition from the described first polynucleotide library.

5. the method for claim 4 comprises in addition:

(k) the described second polynucleotide library is imported the eukaryotic host cell group that can express described immunoglobulin molecules;

(1) imports those polynucleotide that separate from the first polynucleotide library to this host cell;

(m) allow described host cell expression immunoglobulin molecules or its antigen-specific fragment;

(n) this host cell is contacted with described specific antigen; And

(o) those polynucleotide of the coding second immunoglobulin (Ig) subunit polypeptide in the described second polynucleotide library of recovery, the described second immunoglobulin (Ig) subunit polypeptide is special as an immunoglobulin molecules or the segmental part of its antigen-specific for described antigen.

6. the method for claim 5 comprises in addition:

(p) the described polynucleotide that are recovered to are imported the host cell group that can express described immunoglobulin molecules;

(q) import those polynucleotide that separate from the first polynucleotide library to this host cell;

(r) allow host cell expression immunoglobulin molecules or its antigen-specific fragment;

(s) this host cell is contacted with described antigen, and

(t) those polynucleotide of the coding second immunoglobulin (Ig) subunit polypeptide in the described second polynucleotide library of recovery, the described second immunoglobulin (Ig) subunit polypeptide is special as an immunoglobulin molecules or the segmental part of its antigen-specific for described antigen.

7. the method for claim 6, comprise in addition step (p)-(t) is repeated one or repeatedly, thereby the polynucleotide of the coding second immunoglobulin (Ig) subunit polypeptide in the described second polynucleotide library of enrichment, wherein this second immunoglobulin (Ig) subunit polypeptide can be specifically in conjunction with described antigen as an immunoglobulin molecules or the segmental part of its antigen-specific.

8. the method for claim 7 comprises those polynucleotide that separation is recovered in addition from the described second polynucleotide library.

9. the method for preparing segmental first polynucleotide of coding for antigens specific immunoglobulin molecule or its antigen-specific and second polynucleotide will be comprising will be combined according to the method separated coding first immunoglobulin (Ig) subunit polypeptide of claim 8 or segmental first polynucleotide of its antigen-specific and the coding second immunoglobulin (Ig) subunit polypeptide or segmental second polynucleotide of its antigen-specific.

10. the method for preparing antigen expressed specific immunoglobulin molecule or the segmental host cell of its antigen-specific is comprising will expressing in the eukaryotic host cell of described polynucleotide with coding second immunoglobulin (Ig) subunit polypeptide or segmental second polynucleotide importing of its antigen-specific according to coding first immunoglobulin (Ig) subunit polypeptide or segmental first polynucleotide of its antigen-specific that the method for claim 9 is produced.

11. host cell according to the preparation of the method for claim 10.

12. preparation antigen specific immune globulin molecule or the segmental method of its antigen-specific, comprising:

The host cell of cultivation claim 11 under the condition of expression can take place at the described coding first immunoglobulin (Ig) subunit polypeptide or segmental first polynucleotide of its antigen-specific and the coding second immunoglobulin (Ig) subunit polypeptide or segmental second polynucleotide of its antigen-specific; With the described antigen specific immune globulin molecule of recovery or its antigen-specific fragment.

13. the process of claim 1 wherein that described immunoglobulin molecules is the human normal immunoglobulin molecule.

14. the process of claim 1 wherein that the described first immunoglobulin (Ig) subunit polypeptide is a heavy chain immunoglobulin, or its antigen-specific fragment.

15. the method for claim 14, wherein said heavy chain immunoglobulin are the heavy chain immunoglobulins of secreted form.

16. the method for claim 14, the heavy chain immunoglobulin that wherein said heavy chain immunoglobulin or its antigen-specific fragment are film combining form.

17. the method for claim 16, wherein said heavy chain immunoglobulin or its antigen-specific fragment comprise natural immunoglobulin (Ig) membrane spaning domain.

18. the method for claim 16, wherein said heavy chain immunoglobulin or its antigen-specific fragment are as the part of fusion rotein and attached on the described host cell.

19. the method for claim 18, wherein said fusion rotein comprises the allos membrane spaning domain.

20. the method for claim 18, wherein said fusion rotein comprises the fas death domain.

21. the method for claim 14, wherein said heavy chain immunoglobulin or its antigen-specific fragment are selected from the IgM heavy chain, IgD heavy chain, IgG heavy chain, IgA heavy chain, IgE heavy chain, and the antigen-specific fragment of any described heavy chain.

22. the method for claim 21, wherein said heavy chain immunoglobulin or its antigen-specific fragment comprise IgM heavy chain or its antigen-specific fragment.

23. the method for claim 14, wherein said immunoglobulin heavy chain constant region sequence comprises the modification that can support altered immunological effect subfunction.

24. the process of claim 1 wherein that the described second immunoglobulin (Ig) subunit polypeptide is a light chain immunoglobulin, or its antigen-specific fragment.

25. the method for claim 24, wherein said light chain immunoglobulin or its antigen-specific fragment can be united with described heavy chain immunoglobulin or its antigen-specific fragment, thereby produce immunoglobulin molecules or its antigen-specific fragment.

26. the method for claim 24, wherein said light chain immunoglobulin is selected from κ light chain and lambda light chain.

27. the process of claim 1 wherein that the described first polynucleotide library construction is in the eucaryon virus vector.

28. the process of claim 1 wherein that the described second polynucleotide library construction is in the eucaryon virus vector.

29. the method for claim 5, the polynucleotide of wherein said separation from the first polynucleotide library utilize the eucaryon virus vector to import.

30. the process of claim 1 wherein that the described second polynucleotide library construction is in plasmid vector.

31. the method for claim 27, wherein said host cell infects in about 1 to 10 the first polynucleotide library with MOI, and the described second polynucleotide library is imported into allowing each infected host cell can absorb under the condition that reaches 20 polynucleotide in the described second polynucleotide library.

32. the method for claim 5, wherein said separation imports described host cell from the polynucleotide in the first polynucleotide library in plasmid vector.

33. the method for claim 27, wherein said eucaryon virus vector is an animal virus vector.

34. the method for claim 28, wherein said eucaryon virus vector is an animal virus vector.

35. the method for claim 33, wherein said animal virus vector can produce infectious viral particle in mammalian cell.

36. the method for claim 35, the natural gene group of wherein said animal virus vector is DNA.

37. the method for claim 35, the natural gene group of wherein said animal virus vector is RNA.

38. the method for claim 36, the natural gene group of wherein said animal virus vector is a linear dsdna.

39. the method for claim 28, wherein said eucaryon virus vector is an animal virus vector, and this eucaryon virus vector is a linear dsdna.

40. the method for claim 38, wherein said animal virus vector is selected from adenovirus carrier, herpesvirus vector and poxvirus vector.

41. the method for claim 40, wherein said animal virus vector is a poxvirus vector.

42. the method for claim 41, wherein said poxvirus vector is selected from the vaccinia subgroup virus carrier, fowlpox virus carrier, goat capripoxvirus carrier, hare poxvirus carrier, entomopoxvirus carrier and pig pox virus carrier.

43. the method for claim 42, wherein said vaccinia subgroup virus carrier is the raccoonpox virus carrier.

44. the method for claim 42, wherein said host cell allows to produce the infectious viral particle of described virus.

45. the method for claim 40, the transcription regulatory region in the wherein said first polynucleotide library can play a role in by the kytoplasm of the cell of poxvirus infection.

46. the method for claim 30, wherein said plasmid vector instructs the described second immunoglobulin (Ig) subunit synthetic in by the kytoplasm of the cell of poxvirus infection by operably connecting transcription regulatory region.

47. the method for claim 45, wherein said transcription regulatory region comprises promotor.

48. the method for claim 47, wherein said promotor is a composing type.

49. the method for claim 48, wherein said promotor are vaccinia virus p7.5 promotors.

50. the method for claim 48, wherein said promotor are synthetic morning/late promoters.

51. the method for claim 47, wherein said promotor are activated T7 phage promoters in the cell of expressing t7 rna polymerase.

52. the method for claim 45, wherein said transcription regulatory region comprises transcription termination region.

53. the method for claim 38, the wherein said first polynucleotide library construction is in the eucaryon virus vector, and construction process comprises:

(a) the linear DNA viral genome of cutting and separating, thus first viral fragment and second viral fragment produced, and wherein said first fragment and second fragment do not have homology;

(b) provide the transferring plasmid group, these plasmids comprise the described heavy chain immunoglobulin group's that operably links together, encodes with transcription regulatory region described polynucleotide, side joint has 5 ' flanking region and 3 ' flanking region, wherein said 5 ' flanking region and the described first viral fragment homology, the 3 ' flanking region and the second viral fragment homology; And wherein said transferring plasmid can with the described first and second viral fragment homologous recombination, thereby the viral genome of form living;

(c) with the described transferring plasmid and first and second viral fragments, under this transferring plasmid and viral fragment can carry out the condition of homologous recombination in the body, import host cell, thereby produce the adorned live virus genome that contains Nucleotide more than the coding heavy chain immunoglobulin; And

(d) reclaim described adorned viral genome.

54. the method for claim 39, the wherein said second polynucleotide library construction is in the eucaryon virus vector, and construction process comprises:

(a) thus the linear DNA viral genome of cutting and separating produces first viral fragment and second viral fragment, wherein said first fragment and second fragment do not have homology;

(b) provide the transferring plasmid group, these plasmids comprise the described light chain immunoglobulin group's that operably links together, encodes with transcription regulatory region polynucleotide, side joint has 5 ' flanking region and 3 ' flanking region, wherein said 5 ' flanking region and the described first viral fragment homology, the 3 ' flanking region and the second viral fragment homology; And wherein said transferring plasmid can with the described first and second viral fragment homologous recombination, thereby the viral genome of form living;

(c) with the described transferring plasmid and first and second viral fragments, under this transferring plasmid and viral fragment can carry out the condition of homologous recombination in the body, import host cell, thereby produce the adorned live virus genome that contains Nucleotide more than the coding light chain immunoglobulin; And

(d) reclaim described adorned viral genome.

55. the process of claim 1 wherein that the polynucleotide of described coding for antigens specific immunoglobulin molecule are identified by detecting following effect:

(a) necrocytosis of antigen induction;

(b) the signal transmission of antigen induction; Perhaps

(c) antigen-specific combination.

56. the method for claim 5, the polynucleotide of wherein said coding for antigens specific immunoglobulin molecule are identified by detecting following effect:

(a) necrocytosis of antigen induction;

(b) the signal transmission of antigen induction; Perhaps

(c) antigen-specific combination.

57. the method for claim 55, wherein said effect are the necrocytosiss of antigen induction.

58. the method for claim 56, wherein said effect are the necrocytosiss of antigen induction.

59. the method for claim 57, wherein said host cell is at its surface expression immunoglobulin molecules, and these expression can the crosslinked generation of the described immunoglobulin molecules of the former inductive of facedown be replied by the experience apoptosis with the host cell of described antigen bonded immunoglobulin molecules.

60. the method for claim 58, wherein said host cell is at its surface expression immunoglobulin molecules, and these expression can the crosslinked generation of the described immunoglobulin molecules of the former inductive of facedown be replied by the experience apoptosis with the host cell of described antigen bonded immunoglobulin molecules.

61. the method for claim 55, wherein said effect are the signal transmission of antigen induction.

62. the method for claim 56, wherein said effect are the signal transmission of antigen induction.

63. the method for claim 61, wherein said host cell is at its surface expression immunoglobulin molecules, these expression can be with the host cell of described antigen bonded immunoglobulin molecules by expressing detectable reporter molecule to antigen inductive immunoglobulin molecules crosslinked generation reply.

64. the method for claim 62, wherein said host cell is at its surface expression immunoglobulin molecules, these expression can be with the host cell of described antigen bonded immunoglobulin molecules by expressing detectable reporter molecule to antigen inductive immunoglobulin molecules crosslinked generation reply.

65. the method for claim 63, wherein said reporter molecule is selected from luciferase, green fluorescent protein and beta-galactosidase enzymes.

66. the method for claim 64, wherein said reporter molecule is selected from luciferase, green fluorescent protein and beta-galactosidase enzymes.

67. the method for claim 55, wherein said effect are the antigen-specific combinations.

68. the method for claim 67 comprises:

(a) the expressed antigen specific immune globulin molecule of described host cell can with described antigen bonded condition under, the host cell pond is contacted with antigen; With

(b) duplicate the polynucleotide that reclaim the first polynucleotide library the pond from those expression and the host cell pond of described antigen generation bonded immunoglobulin molecules or the polynucleotide of before having reserved.

69. the method for claim 68 comprises in addition:

(c) the described polynucleotide that are recovered to are divided into a plurality of inferior ponds, these inferior ponds are imported the host cell group that can express described immunoglobulin molecules;

(d) allow described host cell expression immunoglobulin molecules or its antigen-specific fragment;

(e) the expressed antigen specific immune globulin molecule of described host cell can with described antigen bonded condition under, described pond is contacted with antigen; And

(f) duplicate the polynucleotide that reclaim the first polynucleotide library the pond from those expression and the host cell pond of described antigen generation bonded immunoglobulin molecules or the polynucleotide of before having reserved.

70. the method for claim 69, comprise in addition step (c)-(f) is repeated one or repeatedly, thereby the polynucleotide of the coding first immunoglobulin (Ig) subunit polypeptide in the described first polynucleotide library of enrichment, wherein this first immunoglobulin (Ig) subunit polypeptide can be specifically in conjunction with described antigen as an immunoglobulin molecules or the segmental part of its antigen-specific.

71. the method for claim 67, wherein said antigen are attached on the matrix, this matrix is selected from synthetic particle, polymkeric substance, the tissue culturing plate of magnetic bead and protein bag quilt.

72. the method for claim 67, wherein said antigen presentation are on the surface of the presenting cell of antigen expressed, the presenting cell of this antigen expressed does not make up by having antigenic presenting cell with the described antigenic polynucleotide transfection of operably encoding.

73. the method for claim 72, the presenting cell of wherein said antigen expressed are structured in the no antigen presenting cell that is selected from down group: L cell, Cos7 cell, 293 cells, HeLa cell and NIH 3T3 cell.

74. the method for claim 68, wherein said antigen has been puted together fluorescence labels, by fluorescence-activated cell sorting identify expression can with the host cell pond of antigen bonded immunoglobulin molecules.

75. the method for claim 56, wherein said effect are the antigen-specific combinations.

76. the method for claim 75 comprises:

(a) the expressed antigen specific immune globulin molecule of described host cell can with described antigen bonded condition under, the host cell pond is contacted with antigen; With

(b) duplicate the polynucleotide that reclaim the second polynucleotide library the pond from those expression and the host cell pond of described antigen generation bonded immunoglobulin molecules or the polynucleotide of before having reserved.

77. the method for claim 76 comprises in addition:

(c) the described polynucleotide that are recovered to are divided into a plurality of inferior ponds, these inferior ponds are imported the host cell group that can express described immunoglobulin molecules;

(d) allow described host cell expression immunoglobulin molecules, or its antigen-specific fragment;

(e) the expressed antigen specific immune globulin molecule of described host cell can with described antigen bonded condition under, described pond is contacted with antigen; And

(f) duplicate the polynucleotide that reclaim the second polynucleotide library the pond from those expression and the host cell pond of described antigen generation bonded immunoglobulin molecules or the polynucleotide of before having reserved.

78. the method for claim 77, comprise in addition step (c)-(f) is repeated one or repeatedly, thereby the polynucleotide of the coding first immunoglobulin (Ig) subunit polypeptide in the described first polynucleotide library of enrichment, wherein this first immunoglobulin (Ig) subunit polypeptide can be specifically in conjunction with described antigen as an immunoglobulin molecules or the segmental part of its antigen-specific.

79. the method for claim 75, wherein said antigen are attached on the matrix, this matrix is selected from synthetic particle, polymkeric substance, the tissue culturing plate of magnetic bead and protein bag quilt.

80. the method for claim 75, wherein said antigen presentation are on the surface of the presenting cell of antigen expressed, the presenting cell of this antigen expressed does not make up by having antigenic presenting cell with the described antigenic polynucleotide transfection of operably encoding.

81. the method for claim 80, the presenting cell of wherein said antigen expressed are structured in the no antigenic presenting cell that is selected from down group: L cell, Cos7 cell, 293 cells, HeLa cell and NIH3T3 cell.

82. the method for claim 76, wherein said antigen has been puted together fluorescence labels, by fluorescence-activated cell sorting identify expression can with the host cell pond of antigen bonded immunoglobulin molecules.

83. the method in preparation coding immunoglobulin (Ig) subunit polypeptide group's polynucleotide library in the eucaryon virus vector, this method comprises:

(a) the linear DNA viral genome of cutting and separating, thus first viral fragment and second viral fragment produced, and wherein said first fragment and second fragment do not have homology;

(b) provide the transferring plasmid group, these plasmids comprise the immunoglobulin (Ig) subunit polypeptide group's that operably links together, encodes with transcription regulatory region polynucleotide group, side joint has 5 ' flanking region and 3 ' flanking region, wherein said 5 ' flanking region and the described first viral fragment homology, the 3 ' flanking region and the second viral fragment homology; And wherein said transferring plasmid can with the described first and second viral fragment homologous recombination, thereby the viral genome of form living;

(c) with the described transferring plasmid and first and second viral fragments, under this transferring plasmid and viral fragment can carry out the condition of homologous recombination in the body, import host cell, thereby produce the adorned live virus gene group that respectively contains Nucleotide more than the coding immunoglobulin (Ig) subunit polypeptide; And

(d) reclaim described adorned virogene cohort.

84. the method for claim 83, wherein each immunoglobulin (Ig) subunit polypeptide comprises:

(a) be selected from first constant region for immunoglobulin of the group of forming by CH and constant region of light chain;

(b) corresponding to the immune globulin variable region of described first constant region; With

(c) can instruct the cell surface expression or the excretory signal peptide of the described first immunoglobulin (Ig) subunit polypeptide.

85. a test kit that is used to be chosen in the antigen-specific recombination immunoglobulin of expressing in the eukaryotic host cell wherein comprises:

(a) operably connect transcription regulatory region, the coding the first immunoglobulin (Ig) subunit polypeptide group the first polynucleotide library, wherein each first immunoglobulin (Ig) subunit polypeptide comprises:

(i) be selected from first constant region for immunoglobulin of the group of forming by CH and constant region of light chain,

(ii) with the corresponding immune globulin variable region of described first constant region, and

(iii) can instruct the cell surface expression or the excretory signal peptide of the described first immunoglobulin (Ig) subunit polypeptide,

The wherein said first polynucleotide library construction is in the eucaryon virus vector;

(b) operably connect transcription regulatory region, the coding the second immunoglobulin (Ig) subunit polypeptide group the second polynucleotide library, wherein each second immunoglobulin (Ig) subunit polypeptide comprises:

(i) be selected from second constant region for immunoglobulin of the group of being made up of CH and constant region of light chain, wherein this second constant region for immunoglobulin is different with first constant region for immunoglobulin,

(ii) with the corresponding immune globulin variable region of described second constant region, and

(iii) can instruct the cell surface expression or the excretory signal peptide of the described second immunoglobulin (Ig) subunit polypeptide,

The wherein said second immunoglobulin (Ig) subunit polypeptide can make up with the first immunoglobulin (Ig) subunit polypeptide, thereby forms immunoglobulin molecules, or its antigen-specific fragment, and this second polynucleotide library construction is in the eucaryon virus vector; With

(c) can express the host cell group of described immunoglobulin molecules;

The wherein said first and second polynucleotide libraries all provide with two kinds of forms of virion of infectious viral particle and deactivation, and the virion of described deactivation energy host cells infected, make it express the first and second immunoglobulin (Ig) subunit polypeptides, but do not carry out virus replication; And

The antigen specific immune globulin molecule of described host cell expression is by selecteed with antigenic interaction.

86. coded antibody or its antigen-specific fragment of polynucleotide that makes by method according to claim 1.

87. composition that comprises described antibody of claim 86 and pharmaceutical acceptable carrier.

88. the method for the antigen specific immune globulin molecule or the segmental polynucleotide of its antigen-specific in the single structure territory of selecting to encode comprises:

(a) the polynucleotide library that will operably connect transcription regulatory region, coding single structure territory immunoglobulin polypeptides group imports in the eukaryotic host cell group that can express described immunoglobulin molecules; Wherein each immunoglobulin polypeptides comprises:

(i) immunoglobulin heavy chain constant region,

The (ii) immunoglobulin heavy chain variable region of camel sourceization, and

(iii) can instruct the cell surface expression or the excretory signal peptide of described immunoglobulin (Ig) subunit polypeptide;

(b) allow described host cell expression immunoglobulin molecules or its antigen-specific fragment;

(c) described immunoglobulin molecules is contacted with antigen; And

(d) from express can with the host cell of described antigen bonded immunoglobulin molecules in reclaim the library polynucleotide.

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