CN1274717C - Immune suppression fusion protein, its coded nucleic acid and application - Google Patents

Abstract

The present invention relates to a novel immunosuppression fusion protein, coding nucleic acid of the fusion protein and application of the fusion protein.

Description

A kind of novel immunosuppression fusion rotein, its coding nucleic acid and uses thereof

Invention field

The invention belongs to molecular biology, immunology and medicinal design field.More particularly, the present invention relates to a kind of novel immunosuppression fusion rotein, its coding nucleic acid and uses thereof.

Background technology

Allosome hematopoietic stem cell transplantation (HSCT) and the organ transplantation of entity internal organs have become one of most effectual way of chronic diseases such as curing various malignant hematologic diseases, heredopathia, severe radiation syndrome and severe combined immunodeficiency up to now and various organ function depletion.Because the difference of tissue compatible complex body (MHC) between confession-receptor (also having other many X factor certainly), graft versus host disease (GVH disease) (GVHD or GVHR) and host versus graft response (HVGD) inevitably will take place, and this is to cause allosome HSCT and organ transplantation one of the most important reason of failing.Therefore, GVHD and HVGD have become the biggest obstacle that allosome HSCT and organ transplantation clinically face.Yet the greatest drawback that the immunosuppression associating measure of the present multiple prevention of having developed and having used and control GVHD and HVGD exists is exactly that targeting specific is poor, the general toxic side effect big, the immunological tolerance phase is short.Therefore, press for and to improve or to overcome the drawback that above-mentioned IM exists, create the special control of a new generation's energy or weaken GVHD and the measure of HVGD.

Generally acknowledge that at present in the cellular factor of numerous participation graft-rejections, CD4+, CD8+T cell are in central status, it needs through three phases from being activated to the performance biological effect.The extracorporeal blocking experiment that this three phases carries out is respectively found, immunosuppressive action has only been played in the blocking-up in first and second stage, and blocking-up phase III B7:CD28/CTLA4 costimulatory signal system (being called for short B7:CD28/CTLA4), the T cell has just produced immune incapability (anergy).For example in primary immune response, no matter using anti-CD28Fab still is that CTLA4-Ig blocking-up B7:CD28/CTLA4 costimulatory signal system all can suppress secondary immunne response significantly.The more important thing is, in primary immune response, only in the presence of B7 molecule, secondary immunne response could take place, illustrate that the B7:CD28/CTLA4 system all plays decisive role in mediation primary immune response or secondary immunne response.

More make the investigator that pleasantly surprised breakthrough is, utilize mouse body inner model can prove the discovery of above-mentioned experiment in vitro fully, promptly after having blocked the B7:CD28/CTLA4 system in the body, then can cause immunological tolerance, not only can make the graft of acceptor mouse withstand long term exposure mouse of the same race, even and can the human antigen of withstand long term exposure xenogenesis.As people's islet cell transplantation is treated down and with novel recombinant C TLA4-Ig to the renal capsule of manual-induced diabetic mice, not only function is normal to found that the human pancreatic island cell of treatment group transplanting, and any histology evidence of transplant rejection do not occur; Get former donor people's islet cell transplantation again and give these mouse, CTLA4-Ig treatment group graft survival time obviously prolongs, and the Ig control group then can not.Prompting CTLA4-Ig can cause the specificity tolerance of host to graft in vivo.The bone marrow transplantation experiment that xenogenesis rat heart is subsequently transplanted and MHC does not match between animal all adds confirmation, under specific environment, CTLA4-Ig can prolong the survival time of graft effectively, and does not need the participation of other immunosuppressor, and the CTLA4-Ig treatment does not also influence the reconstruction of hemopoietic function.The scientist of U.S. Harvard university in 1999 is reported first in the world, use GVHD behind the recombinant C TLA4-Ig blocking-up B7:CD28/CTLA4 control patient of the system hematopoietic cell transplantation and obtain significant curative effect (people such as Guinan, N.Engl.J.Med.1999,340:1704-1714), thus established solid basis for the more further investigation of this strategy especially clinical application.Therefore, no matter the B7:CD28/CTLA4 system is for inducing immune tolerance or the most key aspect the control GVHD, is just becoming the focus that current allosome hematopoietic stem cell transplantation, transplantation immunity, organ transplantation and relevant neotype immunosuppressant novelty development research field are paid close attention to the most.

Yet, make a general survey of the research means of exploring at present control GVHD of B7:CD28/CTLA4 system or inducing immune tolerance in the world, employing all be with anti-B7 monoclonal antibody/how anti-or CTLA4-Ig seals or blocks.Its defective is: the first, and no matter be anti-B7 monoclonal antibody/how anti-or the sealing of CTLA4-Ig or blocking-up, the inductive immunological tolerance time limit is too short; The second, only seal the inductive T of institute of B7:CD28/CTLA-4 system cellular immunization tolerance can transplanted postoperative infection etc. IL-2 and other cytokine of release that factor causes reverse, cause graft failure; The 3rd, anti-B7 monoclonal antibody/resist used when sealing or blocking-up is mouse source property more, and toxic side effect is big.Therefore, still need new immunosuppressor, so that more effectively suppress immunological rejection, the treatment autoimmune disease.

One object of the present invention is to provide a kind of fusion rotein, the syzygy that wherein contains immune aglucon or its functional fragment or derivative and toxin protein, can selectively targeted lymphocyte, thus selective killing participates in the lymphocyte of transplant rejection or autoimmune disease.

The invention summary,

Be well known in the art, have very high binding specificity between the corresponding aglucon of acceptor with it.The contriver utilizes this specificity to come the toxin protein that the target importing can killer cell, also kills relevant lymphocyte in selective exclusion immune system activation approach, thereby reaches the effect of specificity control GVHD and inducing immune tolerance.

One aspect of the present invention provides a kind of fusion rotein, wherein comprises the syzygy of immune aglucon and toxin protein.

The present invention provides the polynucleotide of encoding said fusion protein on the other hand.

The present invention also provides nucleic acid construct, expression vector, the host cell that contains described polynucleotide.

The present invention also provides production method of described fusion rotein and uses thereof.

Description of drawings

Fig. 1 is the electrophorogram of B7-2 and B7-1 extracellular region cDNA fragment PCR amplified production.M is a DL2000PCR molecular weight sign, and swimming lane 1 is a B7-1 cDNA extracellular region amplified production; Swimming lane 2 is a B7-2 cDNA extracellular region amplified production.

Fig. 2 is the structural representation of pRSET-B7-2-PE40KDEL recombinant plasmid.

Fig. 3 is the full product of cell lysis SDS-PAGE electrophoresis result that contains the host cell BL-21-CodonPlus-RIL of pRSET-B7-2-PE40KDEL recombinant plasmid.M1 is a molecular weight protein marker.Swimming lane 1 is the full product of cell lysis of inductive not, and swimming lane 2 and 3 is the full product of cell lysis of inductive.

Fig. 4 is for expressing thalline BL21 (DE3) pLysS/pRSETA-B7-2-DE40KDEL fusion rotein content scanning result.

Fig. 5 is the Western engram analysis of pRSETA-B7-2-DE40KDEL recombinant vectors expression.M2 is a molecular weight protein marker; Swimming lane 4 is the full product of cell lysis of inductive not; Swimming lane 5 and 6 is the full product of cell lysis of inductive.

Fig. 6 is the cell toxicant killing activity figure of reorganization fusion toxin protein B 7-2-PE40KDEL to Jurkat and Raji clone.

The sequence explanation

SEQ ID NO:1 and 2 is respectively nucleotide sequence and the amino acid sequence corresponding that recombinant immune suppresses syzygy B7-2-PE40KDEL.

SEQ ID NO:3 and 4 nucleotide sequence and amino acid sequence corresponding for reorganization immunosuppression syzygy B7-1-PE40KDEL.

SEQ ID NO:5 is the sequence of forward primer PF7-2A.

SEQ ID NO:6 is the sequence of reverse primer PR7-2A.

SEQ ID NO:7 is the sequence of forward primer PF7-1A.

SEQ ID NO:8 is the sequence of reverse primer PR7-1A.

SEQ ID NO:9 and 11 is respectively the nucleotide sequence and the amino acid sequence corresponding of B7-2 extracellular region.

SEQ ID NO:10 and 12 is respectively the nucleotide sequence and the amino acid sequence corresponding of B7-1 extracellular region.

SEQ ID NO:13 is the sequence of forward primer PF7-2B.

SEQ ID NO:14 is the sequence of reverse primer PR7-2B.

SEQ ID NO:15 is the sequence of forward primer PF7-1B.

SEQ ID NO:16 is the sequence of reverse primer PR7-1B.

SEQ ID NO:17 is the sequence of forward primer PF-PEA.

SEQ ID NO:18 is the sequence of reverse primer PR-PEA.

SEQ ID NO:19 is the sequence of reverse primer PR-PEB.

Detailed Description Of The Invention

The present invention relates to a kind of fusion, wherein comprise the fusion of immune aglucon and toxin protein.

In the present invention, term " immune aglucon " refers to energy and lymphocyte, combine such as the cell receptor on the T lymphocyte, participate in the molecule that immune response pathway such as the second signal system activate, comprise natural aglucon or its variant, derivative or part, they have kept the ability that combines with corresponding cell receptor basically.

In one embodiment, the corresponding acceptor of described immune aglucon is present on the part or all of lymphocyte. More preferably, described aglucon is B7 such as B7-1, B7-2, B7-3, BB1 or CD40L etc. Further, described aglucon is B7, and it can combine with cell receptor CD28.

Randomly, described aglucon is B7-1. Equally preferably, described aglucon is B7-2. It can derive from mammal, comprises people, mouse, rat etc.

Immune aglucon in the fused protein of the present invention can be full-length proteins, or its fragment, derivative or variant etc., and they have kept desirable purpose activity basically, refer to herein the activity that combines with corresponding acceptor. In one embodiment, the immune aglucon in the fusion is the fragment that comprises its ectodomain.

Derivative can be following each peptide species: (i) wherein one or more amino-acid residues are replaced by conservative or nonconservative amino-acid residue (preferred conservative amino-acid residue), or (ii) wherein other amino acid merges with it mutually, as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of purified polypeptide.In view of the teachings contained herein, those skilled in the art are not difficult to grasp this fragment and derivative.

Some aminoacid sequences that it should be recognized by those skilled in the art that immune aglucon polypeptide can change, and can proteinic structure of remarkably influenced or function.If this species diversity in the analytical sequence should be able to remember to exist in the protein the active vital zone of decision.Generally speaking, can replace the residue that forms tertiary structure, as long as use the residue of exercising similar functions.Under other occasion, occur in proteinic non-important area if change, the type of residue might be inessential fully so.

Therefore, the present invention comprise in addition show basically can with the various varients and the fragment of the immune aglucon of corresponding cell receptors bind.This varient comprises that disappearance, insertion, inversion, repetition and type replace (as principle is to replace another kind of hydrophilic residue with a kind of hydrophilic residue, rather than replaces strong hydrophobic residue with the residue of strong hydrophilicity).Little variation or this " neutral " aminoacid replacement are generally very little to activity influence.

Hereinafter is the example that conserved amino acid well known to those skilled in the art replaces:

Aromatic series: phenylalanine, tryptophane, tyrosine

Hydrophobic: leucine, Isoleucine, Xie Ansuan

Polar: glutamine, l-asparagine

Alkalescence: arginine, Methionin, Histidine

Tart: aspartic acid, L-glutamic acid

Little: L-Ala, Serine, Threonine, methionine(Met), glycine

Being used for toxin protein of the present invention can be the protein that plant poison such as pair cells such as Ricin, phytohemagglutinin, toxalbumin or bacteriotoxin have kill capability, and bacteriotoxic example includes but not limited to diphtheria toxin, shiga toxin, Pseudomonas exotoxin such as Pseudomonas aeruginosa extracellular toxin etc.

In the context of the invention, term " toxin protein " not only comprises native protein, also comprises its variant, derivative and fragment etc., and they have and the essentially identical cell killing activity of parent's albumen.The definition of relevant " variant ", " derivative ", " fragment " is referring to the content about " immune aglucon ".

In fusion rotein of the present invention, immune aglucon can directly be connected with toxin protein.Preferably, they link to each other by comprising one or more amino acid whose connection peptides.This connection peptides can have 1-100 amino acid, preferably has 1-50,1-25,1-20,1-15, a 1-10 or 1-5 amino acid.Further preferably, described connection peptides is (Gly ₄Ser) ₄

Fusion rotein of the present invention can be through genetically engineered production, also can chemosynthesis.Perhaps, in the fusion rotein of the present invention, can there be one or more amino-acid residues to comprise substituting group.In addition, fusion rotein of the present invention can also and additional compounds, be connected as the compound (as polyoxyethylene glycol) that improves the polypeptide half life.These derivatives include within the scope of the present invention.

Polynucleotide

The present invention also comprises the polynucleotide of encoding said fusion protein, and wherein encoding sequence can merge with the polynucleotide sequence that helps host cell expression and secrete polypeptide (for example transporting the leader sequence of polypeptide as the secretion sequence functionating with control from transit cell) in identical reading frame.

Polynucleotide of the present invention also can have the encoding sequence that merges with a kind of flag sequence in frame, described flag sequence can be used for purifying polypeptide of the present invention.The host is under the situation of bacterium, this flag sequence can be the mature polypeptide that is merged with purifying and flag sequence by the six histidine mark things that the pQE-9 carrier provides, or for example, when using mammalian hosts, during as the COS-7 cell, flag sequence also can be hemagglutinin (HA) marker.The HA marker is equivalent to derive from the proteic epi-position of influenza virus hemagglutinin (Wilson, I etc., cell, 37:767 (1984)).

Carrier and host cell

The present invention also relates to comprise the carrier of polynucleotide of fusion rotein of the present invention of encoding, the method for carrying out genetic engineering modified host cell and producing fusion rotein by recombinant technology with recombinant vectors.

With carrier of the present invention host cell is carried out genetic engineering modified (transduction or conversion or transfection), described carrier can be for example cloning vector or expression vector.The form of carrier can be a plasmid for example, virion, phage or the like.

Polynucleotide of the present invention can be used to produce polypeptide by recombinant technology, therefore, for example, described polynucleotide can be included in in the multiple expression vector any with express polypeptide.This carrier comprises chromosomal, achromosomal and the synthetic dna sequence dna, as the derivative of SV40; Bacterial plasmid; Phage DNA; Baculovirus; Yeast plasmid; Carrier derived from the associating of plasmid and phage DNA, viral DNA (as vaccinia virus, adenovirus, fowlpox virus and pseudorabies virus).Yet as long as can duplicate in the host and survive, any other carrier all can use.

Dna sequence dna in the expression vector links to each other synthetic to mediate it with suitable expression control sequenc (promotor) effectively.As for the representative example of this promotor, that should mention has: LTR or SV40 promotor, intestinal bacteria lac or trp, phage P _LPromotor and known other promotor that can control the genetic expression in prokaryotic cell prokaryocyte or eukaryotic cell or their virus.Expression vector also contains ribosome bind site and the transcription terminator that is useful on initial translation.Carrier also comprises the suitable sequence that is used to strengthen expression.

In addition, expression vector preferably contains one or more selected markers and can be used for selecting the phenotypic character of transformed host cells to provide, as be used for the Tetrahydrofolate dehydrogenase or the neomycin resistance of eukaryotic cell culture or tsiklomitsin in the intestinal bacteria and amicillin resistance.

Can use and contain suitable dna sequence dna mentioned above and suitable promotor or the carrier of control sequence and transform suitable host so that host expresses protein.

As for suitable host's representative example, that should mention has: bacterial cell, and as intestinal bacteria, streptomycete, Salmonella typhimurium; The fungal cell is as yeast; Insect cell is as fruit bat S2 and Spodoptera Sf9; Zooblast, as CHO, COS or Bowes melanoma cells; Adenovirus; Vegetable cell etc.According to the instruction of this paper, those skilled in the art should be able to grasp how to select suitable host.

Use also can utilize acellular translation system to produce this protein derived from the RNA of DNA construct of the present invention.The suitable clone and the expression vector that can use in protokaryon and eucaryon host are described in people such as Sambrook, molecular cloning: laboratory manual, and the 2nd edition, the cold spring port, New York (1989), the document has been listed this paper in as a reference.

Can increase higher eucaryotic cells transcribing by in carrier, inserting enhancer sequence to the DNA of code book invention polypeptide.Enhanser is that promotor is worked to increase the DNA element of its cis acting of transcribing, and example comprises sub-enhanser of cytomegalovirus early promoter and adenovirus enhanser etc.

For will the translation protein secreting in endoplasmic, outer pericentral siphon space or born of the same parents' external environment, suitable secretion signal can be mixed by in the polypeptide expressed.

Polypeptide can be modified form expressed.Polypeptide not only can comprise secretion signal, also can comprise other allos functional area.For example, can add other amino acid region at the N-of polypeptide end, especially charged amino acid is to improve polypeptide in host cell, in the purge process or subsequently processing and stability in the storage process and persistence.Also can in polypeptide, add the peptide moiety so that purifying finally prepares polypeptide and can remove this class zone before.Those skilled in the art know in polypeptide add the peptide moiety with cause secretion or outside secrete, improve stability and be convenient to purifying or the like, this is the routine techniques in this area.

Transform the appropriate host bacterial strain and host strain is cultured to after the suitable cell density, induce selected promotor by suitable mode (as temperature variation or chemical induction), and cell is cultivated for some time again.

General by centrifugal cell harvesting, by physics or chemical process smudge cells, the crude extract that keeps gained is to be further purified.

By comprising freeze thaw circulation, supersound process, Mechanical Crushing, or use lysis agent etc. can broken be used for the microorganism cells of marking protein in interior any facilitated method, these methods are well-known to those skilled in the art.

Can use multiple mammalian cell culture system to come express recombinant protein, the example of mammalian expression system comprises Gluzman, cell, the described monkey kidney of 23:175 (1981) inoblast COS-7 system, and other clone that can express the capacitive carrier, as C127,3T3, CHO, HeLa and bhk cell system.Mammalian expression vector can contain replication orgin, suitable promotor and enhanser and any essential ribosome bind site, polyadenylation site, donor splicing site and acceptor site, the non-transcribed sequence of transcription termination sequence and 5 ' flank.Can use the dna sequence dna that derives from SV40 montage and polyadenylation site that required non transcribed genetic elements is provided.

By comprising ammonium sulfate or ethanol sedimentation, acid extraction, positively charged ion or anion-exchange chromatography, the phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, the method for hydroxyapatite chromatography and lectin chromatography etc. reclaims from the reconstitution cell culture and purifying fusion rotein of the present invention.At last, can use high performance liquid chromatography (HPLC) to finish final purification step.

Polypeptide of the present invention can be the product of chemical synthesis process, or is produced by recombinant technology by protokaryon or eucaryon host (as the bacterium in cultivating, yeast, higher plant, insect and mammalian cell).Depend on host used in the recombinant method for production, fusion rotein of the present invention can be glycosylated, also can be nonglycosylated.Fusion rotein of the present invention also can comprise initial methionine residues.

Fusion rotein of the present invention or polynucleotide can be used for suppressing over-drastic or unnecessary immune response.In one embodiment, described over-drastic or unnecessary immune response are that graft versus host repels or host versus graft is repelled.In another embodiment, described immune response is an autoimmune disorder, includes but not limited to rheumatoid arthritis, lupus erythematosus, psoriasis, ankylosing spondylitis, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes etc.

Pharmaceutical composition

Polypeptide of the present invention and suitable pharmaceutical carrier can be united use to make pharmaceutical composition.This composition contains fusion rotein of the present invention and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.Described carrier includes but not limited to salt solution, buffer salt solution, dextran, water, glycerine, ethanol and associating thereof.The pattern that preparation should be fit to use.

Fusion rotein of the present invention can be co-administered with the acceptable vehicle of one or more pharmacy in pharmaceutical composition.Should understand when being applied to people patient, the nursing doctor can determine pharmaceutical composition of the present invention total consumption of every day in correct medical judgment scope.Any concrete patient's particular treatment effective dose level depends on multiple factor, comprises the type and the degree of the reaction of desiring to reach, the specific combination of used other medicament (if any); Patient's age, body weight, healthy state, sex and diet; The time of using, the speed that approach of using and composition are secreted outward; The time limit of treatment; With particular composition associating or the medicine (as chemotherapeutic) that uses simultaneously; With the well-known other factors of medical field.Suitable preparation known in the art sees Remington ' s Pharmaceutical Sciences (latest edition), Mack publishing company, Easton, PA.

Consider each patient's clinical condition, the site of release, the method for using, the plan of using and the known other factors of actually operating personnel, prepare and take the used fusion protein compositions of treatment in the mode consistent with good medical practice.Therefore, can be identified for the fusion rotein of " significant quantity " of this paper purpose by above-mentioned consideration.

Mode that can be easy, as the part, intravenously, intraperitoneal, intramuscular, intra-arterial, subcutaneous, in the nose or intradermal routes use described pharmaceutical composition.The amount of institute's drug administration composition should be able to treat and/or prevent over-drastic or unnecessary immune response effectively.

Fusion rotein of the present invention also can be used by expressing in vivo, normal " gene therapy " said that Here it is.

Therefore, for example, can exsomatize and the cell from the patient be transformed, then the cell through transforming be offered the patient who needs with this fusion rotein treatment with the polynucleotide (DNA or RNA) of code book invention fusion rotein.This method is a method known in the art.For example, can utilize the retroviral particle pair cell of the RNA that contains code book invention fusion rotein to transform by method well known in the art.

Similarly, can be by method well known in the art for example, engineered cells is with expressed fusion protein in vivo in vivo.Known in this area, can use producer's cell of the retroviral particle that can produce the RNA that contains code book invention fusion rotein with engineered cells and express polypeptide in vivo in vivo to the patient.In view of instruction of the present invention, those skilled in the art can grasp these and other method that is used for using fusion rotein of the present invention easily.For example, the expression vector that is used for engineered cells not only can be a retrovirus, also can be adenovirus, can be used for engineered cells in vivo after described adenovirus and the associating of suitable transmission carrier.Other example that transmits carrier comprises the carrier system based on HSV, gland relevant viral vector and inert carrier, as be coated with the iron particle of dextran.

The retrovirus that can therefrom obtain retroviral plasmid vector mentioned above includes but not limited to the Moloney murine leukemia virus, spleen necrosis virus, as the Rous sarcoma virus, the Harvey sarcoma virus, avian leukosis virus, gibbon ape leukemia virus (gibbon ape leukemiavirus), the human immunodeficiency virus, adenovirus, bone marrow proliferative sarcoma virus, and mammary tumour virus.In one embodiment, retroviral plasmid vector derives from the Moloney murine leukemia virus.

Carrier comprises one or more promotors, and operable suitable promotor includes but not limited to retroviral LTR; The SV40 promotor; And human cytomegalic inclusion disease virus (CMV) promotor (Miller etc., biotechnology, the 7th volume, 9:980-990 (1989)) or any other promotor (cell promotor for example, as include but not limited to histone, the eukaryotic cell promotor of polIII and beta-actin promotor etc.).Spendable other viral promotors includes but not limited to adenovirus promoter, thymidine kinase (TK) promotor and B19 parvovirus promotor.In view of the teachings contained herein, the selection of suitable promotor is conspicuous to those skilled in the art.

The nucleotide sequence of code book invention fusion rotein is subjected to the control of suitable promotor.Spendable suitable promotor includes but not limited to adenovirus promoter, as adenovirus major late promoter; Or allogeneic promoter, as cytomegalovirus (CMV) promotor; Respiratory syncytial virus (RSV) promotor; Inducible promoter is as MMT promotor, metallothionein promoter; The heat shock protein(HSP) promotor; Albumin promoter; The ApoAI promotor; People's globin promotor; Viral thymidine kinase promoter is as the herpes simplex virus thymidine kinase promotor; Retrovirus LTR (comprising modified retrovirus LTR mentioned above); The beta-actin promotor; With the human growth hormone promotor.Promotor also can be a natural promoter.

Can use retroviral plasmid vector transduction package cell line to form producer's clone.The example of package cell line that can be transfected includes but not limited to PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψ CRE, ψ CRIP, GP+E-86, GP+envAm12 and Miller, the people's gene therapy, the clone described in the 1:5-14 (1990) (listing this paper in as a reference in full).Carrier can be by any method transduction packing cell known in the art.Described method includes but not limited to electroporation, the application of liposome, and CaPO ₄Precipitation.Perhaps also retroviral plasmid vector can be wrapped in the liposome, or with the lipid coupling, be applied to the host then.

Producer's clone produces the infectious retroviral vector particle of the nucleotide sequence that contains code book invention fusion rotein.Can use this retroviral vector particle in vivo or external transduction eukaryotic cell then.Can express the nucleotide sequence of code book invention fusion rotein through the eukaryotic cell of transduction.

The preparation method

The invention still further relates to the method for preparing fusion rotein of the present invention, this method comprises that (a) cultivates host cell under the condition that helps described fusion rotein generation; (b) reclaim this fusion rotein.

In preparation method of the present invention, with means known in the art culturing cell in the nutritional medium that is fit to the polypeptide generation.For example, can be in suitable medium, allowing under expression of polypeptides and/or the isolating condition, come culturing cell by shake-flask culture, laboratory or industrial fermentation jar middle and small scale or large scale fermentation (comprise continuously, in batches, batch charging or solid state fermentation).In the suitable medium that comprises carbon and nitrogenous source and inorganic salt, adopt step known in the art to cultivate.Suitable medium can be provided or can be prepared with reference to disclosed the composition (for example, described in the catalogue of American type culture collection) by supplier.If polypeptide is secreted in the substratum, then can directly from substratum, reclaim polypeptide.If polypeptide is not secreted, can from cell lysate, reclaim.

Can reclaim the polypeptide that is produced with means known in the art.For example, can from substratum, reclaim polypeptide by routine operation (including, but are not limited to centrifugal, filtration, extracting, spraying drying, evaporation or precipitation).

Can come purifying many fusion roteins of the present invention by various operations known in the art, these operations comprise, but (for example be not limited to chromatography, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing and size exclusion chromatography), electrophoresis (for example, the isoelectric focusing of preparation property), differential solubleness (for example ammonium sulfate precipitation), SDS-PAGE or extracting is (referring to for example, protein purification, J.C.Janson and Lars Ryden compile, VCH Publishers, New York, 1989).

Hereinafter further set forth the present invention, but and do not mean that limitation of the present invention in illustrational mode.

Embodiment

The clone of embodiment 1:B7-2 extracellular region cDNA

Delivered B7-2 and B7-1 gene order according to document, designed the primer of amplification coding B7-2 extracellular region 225aa and B7-1 extracellular region 208aa cDNA respectively, and verified through GOLDKEY software.The primer sequence of B7-2 is: forward primer PF7-2A is 5 '-GCGGATCCGCTGCTCCTCTG-3 ' (SEQ ID NO:5), introduces the BamHI site at 5 ' end; Reverse primer PR7-2A is 5 '-CCCGGGTTAAGGAATGTGGTCTGG-3 ' (SEQ ID NO:6), introduces terminator codon TAA and SmaI site at 3 ends.The B7-1 primer is: forward primer PF7-1A is 5 '-GCGGATCC-3 ' (SEQ ID NO:7), introduces the BamHI site at 5 ' end; Reverse primer PR7-1A is 5 '-CCCGGGTTA-3 ' (SEQ ID NO:8), introduces terminator codon TAA and SmaI site at 3 ' end.Primer PF7-2A and PR7-2A and PF7-1A and PR7-1A be amplification coding people B7-2 and B7-1 extracellular region variable region 1-225aa and 1-208aa cDNA fragment respectively, and length estimates to be about respectively 675bp and 624bp.

To contain B7-2 ₂₂₅The pGEM-T-B7-2 of extracellular region cDNA ₂₂₅(people such as Zhang Huili, Chinese applied physiology magazine, 2001,17 (2): 117-120) or B7-1 ₂₀₈The pGEM-TB7-1 of extracellular region cDNA ₂₀₈Recombinant plasmid (people such as Zhang Huili, Chinese applied physiology magazine, 2001,17 (2): 117-120) be template, utilize primer PF7-2A and PR7-2A or PF7-1A and PR7-1A, increase through conventional PCR.Reaction conditions be 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1.5 minutes, 25 circulations were extended 10 minutes for back 72 ℃.1% agarose gel electrophoresis is identified amplification.Referring to Fig. 1.

To connect into pGEM-T carrier (Promega company) behind the pcr amplification product purifying, transformation receptor bacterium JM109, the picking positive recombinant is called pGEM-T-B7-2 ₂₂₅(contain B7-2 ₂₂₅Extracellular region) and pGEM-T-B7-1 ₂₀₈Recombinant plasmid vector (contains B7-1 ₂₀₈Extracellular region), insertion fragment is wherein carried out sequencing.

The result shows that B7-2 extracellular region sequence (SEQ ID NO:9) overwhelming majority except that indivedual bases change is consistent with the GenBank sequence, and B7-1 extracellular region sequence (SEQ ID NO:11) is in full accord with the GenBank sequence.Its amino acid sequence corresponding is shown in SEQ ID NO:10 and 12 respectively.

Embodiment 2: novel recombinant immune suppresses the structure of toxin fusion rotein B7-PE40KDEL expression vector

According to B7-2 ₂₂₅, B7-1 ₂₀₈With the PE40 target-gene sequence, the design B7-2 that can increase ₂₂₅, B7-1 ₂₀₈Forward and reverse primer PF7-2B and PR7-2B, PF7-1B and PR7-1B and PF-PEA and PR-PEA with PE40.In order to construct, make B7-2 ₂₂₅, B7-1 ₂₀₈And between the PE40KDEL gene with flexible joint (Gly ₄Ser) ₄Be connected; On this basis, designed reverse primer PR-PEB again, the difference of PR-PEB and PR-PEA be can make its 5 ' REDLK be replaced into KDEL, and in primer PF7-2B and PF7-1B, introduce EcoRI and MunI restriction enzyme site, the tumor-necrosis factor glycoproteins that comprises one section primer PR7-2B and PR7-1B in primer PF-PEA is introduced SmaI restriction enzyme site and connection peptides [(Gly in primer PR-PEA ₄Ser) ₄] encoding sequence.See table 1 for details.

Table 1. makes up the primer sequence that contains coding B7-PE40KDEL fusion gene

Table 1 Sequence of oligonucleotides for B7-PE40KDEL amplificationB7-2 ₂₂₅：

PF7-2B 5’-GCT GCT CCT CTG AAG ATT CAA G-3’(SEQ ID NO：13)

PR7-2B 5’-CGA CCC ACC ACC GCC CGA GCC ACC GCC ACC GCT TCC ACCGCC TCC AGA TCC GCC GCC ACC AGG AAT GTG GTC TGG 3’(SEQ ID NO：14)

B7-1 ₂₀₈

PF7-1B 5’-CGCGAATTCATG GTTATCCACGTGACC-3’(SEQ ID NO：15)

PR7-1B 5’-CGA CCC ACC ACC GCC CGA GCC ACC GCC ACC GCT TCC ACC GCC TCC AGA TCC GCC GCC ACCGTT ATC AGG AAA ATG CTC TTG C-3’(SEQ ID NO：16)

PE40：

PF-PEA 5’-TCG GGC GGT GGT GGG TCG GGC GGC AGC CTG GCC GCG CTG A-3’(SEQ ID NO：17)

PR-PEA 5’-TCC CCC GGG GGA TTA CTT CAG GTC CTC GCG CGG CGG TTT G-3’(SEQ ID NO：18)

With PE40REDLK rite-directed mutagenesis one-tenth → PE40KDEL:

PR-PEB 5’-TCC CCC GGG GGA TTA CAG CTC GTC CTT CGG CGG TTT GCC GGG CTG GCT-3’(SEQ ID NO：19)

Respectively to contain B7-1 ₂₀₈Or B7-2 ₂₂₅The pGEM-T-B7-2 of extracellular region ₂₂₅Or pGEM-T-B7-1 ₂₀₈Recombinant plasmid vector is a template, and unite the recombinant plasmid vector pZX-PEA (people such as Zheng Liyan who contains Pseudomonas aeruginosa extracellular toxin PE40 gene, China's microbiology and Journal of Immunology, 2001,21 (1): 95-98), utilize primer PF7-2B and PR7-2B, PF7-1B and PR7-1B and PF-PEA and PR-PEA, make up desired B7-PE40KDEL fusion gene by overlapping extension-PCR (SOE-PCR) strategy.This need carry out four PCR reactions.

PCR reaction for the first time is to be template with plasmid pGEMT-B7-2, is forward and reverse primer with PF7-2B and PR7-2B, and the B7-2cDNA that is increased is the complete extracellular region at its N end disappearance 16aa, and its length fragment is 675bp, and makes its 5 ' end have joint.

PCR reaction for the second time is to be template with plasmid pZX-PEA, with PF-PEA and PR-PEA is forward and reverse primer, make the PE40DNA that increased for deletion cell combined function district Ia but comprise the toxin gene in complete function II, III district and Ib district, its length fragment is 1083bp, and makes its 3 ' end have joint.It should be noted that because PE40 gene GC content is higher, reaction system need add certain density dimethyl sulfoxide (DMSO) or glycerine in the gene amplification process, can make the amplified production specificity high.Comprise in the 100ul volume PCR reaction system: 100ng dna profiling, 10 * reaction buffer, each 10pmol of upstream and downstream primer, 10mmol/l dNTP 10ul, 25mmol/l MgSO46ul, dimethyl sulfoxide (DMSO) 10ul, Taq archaeal dna polymerase 2U, distilled water are supplied volume 100ul.The PCR reaction conditions is: 98 ℃ of 3min, loop parameter: 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1.5min, back, the 72 ℃ of extension 10min of 25 circulations.

In PCR reaction for the third time, be the upstream and downstream primer with PF7-2B, PR-PEA, former twice PCR product is a template, amplification is built into the B7-2-PE40 fusion gene.

Carry out the 4th PCR reaction at last, be forward and reverse primer wherein with PF7-2B, PR-PEB, with the B7-2-PE40 fusion gene is template, make carboxyl terminal last 5 amino acid REDLK in PE40 function III district be replaced as endoplasmic reticulum albumen and be detained sequence KDEL by fixed point, carry out the EcoRI/SmaI double digestion after the PCR reaction product reclaims and insert the pGEM-T carrier, the final B7-2-PE40KDEL target gene fragment length that obtains is 1.8kb.The structure of B7-1-PE40KDEL is also taked above-mentioned same policy, and the PCR reaction product reclaims the gene that back MunI/SmaI double digestion also can downcut about 1.8kb fragment length.

To connect into pGEM-T carrier (Promega company) behind above-mentioned B7-1-PE40KDEL that builds and the B7-2-PE40KDEL series fusion gene pcr amplification product purifying, transformation receptor bacterium JM109, the picking positive recombinant is called pGEM-T-B7-1-PE40KDEL and pGEM-T-B7-2-PE40KDEL genophore.Insertion fragment is wherein carried out sequencing, the result shows that two fusion genes are except that some sequence change takes place in the only a few site, the overwhelming majority is in full accord with bibliographical information, the nucleotide sequence of B7-2-PE40KDEL and B7-1-PE40KDEL is referring to SEQ ID NO:1 and 3, and its amino acid sequence corresponding is shown in SEQ ID NO:2 and 4 respectively.

There are 4 amino acid that change has taken place in the primary structure of recombinant toxin fusion protein B7-2-PE40KDE, wherein have 2 may be relevant with the change of secondary structure, secondary structural change as the PE40 district is that 15 amino acid that open the beginning in the PE40-II functional zone become the α spiral by the β lamella, and the most last 3 amino acid in PE40-III district then become βZhuan Jiao by the β lamella.

The primary structure of B7-1-PE40KDEL has 3 amino acid to change, and 15 initial amino acid of PE40-II functional zone become the α spiral by the β lamella in the secondary structure, and the most last 3 amino acid of PE40-III functional zone become βZhuan Jiao by the β lamella.Amino acid change in the primary structure, have only one may be relevant with the change of secondary structure, certainly do not get rid of the possibility that flexible joint has influenced secondary structure yet.

Table 2.1. B7-2-PE40KDEL fusion rotein and B7-2 and PE40KDEL albumen I and II texture ratio are

Table 2.1 primary and second structure comparison among B7-2-PE40KDEL recombinant fusionprotein，B7-2 and PE40KDEL

B7-2-PE40KDEL
			Structure	Primary structure	Secondary structure
B7-2-225 (1-225aa) Link-20 (226-245aa) PE40-II-112 (246-357aa) PE40-Ib-35 (358-392) PE40-III-215 (393-607)	16aa aspartic acid → glutamic acid 246aa glycine → serine 265aa phenylalanine → serine 284aa phenylalanine → serine	Do not change 245-259aa 15 β lamellas → 15 α spirals and do not have the 605-607aa of change 3 β lamellas → 3 β-bends

Table 2.2. B7-1-PE40KDEL fusion rotein and B7-1 and PE40KDEL albumen I and II texture ratio are

Table 2.2 primary and second structure comparison among B7-1-PE40KDEL recombinant fusionprotein，B7-1 and PE40KDEL

B7-1-PE40KDEL
			Structure	Primary structure	Secondary structure
B7-1-208 (1-208aa) Link-20 (208-228aa) PE40-II-112 (229-340) PE40-Ib-35 (341-375) PE40-III-215 (376-590)	181aa methionine → threonine 229aa glycine → serine 356aa alanine → serine	Do not change 229-243aa 15 β lamellas → 15 α spirals and do not have 3 β lamellas → 3 β-bends of change

*: aa.amino acid, amino acid; II, Ib and III are functional domains of PE40 respectively, II, Ib and III are respectively the PE40 functional domain.

Embodiment 3: the expression of recombinant toxin protein fusion gene B7-PE40KDEL

Being template with plasmid pGEM-T-B7-2-PE40KDEL and the pGEM-T-B7-1-PE40KDEL that makes up among the embodiment 2 respectively, is primer amplification purpose fragment with PF7-2B or PF7-1B and PR-PEB.100ul volume PCR reaction system is: 100ngDNA template, 10 * reaction buffer, each 10pmol of upstream and downstream primer, 10mmol/l dNTP 10ul, 25mmol/l MgSO4 6ul, dimethyl sulfoxide (DMSO) 10ul, Taq archaeal dna polymerase 2U, distilled water are supplied volume 100ul.The PCR reaction conditions is: 98 ℃ of 3min, and loop parameter: 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1.5min, after 25 circulations, 72 ℃ are extended 10min.B7-1-PE40KDEL DNA and B7-2-PE40KDEL length fragment that the PCR reaction is increased are respectively 1842bp and 1791bp.The PCR reaction product is inserted the pGEM-T carrier respectively after reclaiming, the BamHI/SmaI double digestion downcuts about 1.8kb fragment, its orientation is inserted in expression vector pRSETA (Invitrogen company), and transform bacterial strain BL21 (DE3) pLysS (Invitrogen company), obtained to express the pRSETA-B7-2-PE40KDEL-expression vector of B7-2-PE40KDEL toxin protein fusion rotein.Its synoptic diagram is seen Fig. 2.

Host cell BL21 (DE3) pLysS that will comprise the pRSETA-B7-2-PE40KDEL recombinant expression vector is cultured to logarithmic phase mid-term in bacterial growth cycle in 37 ℃, after OD600nm is about 0.2-0.5, with 0.1mM IPTG in 22-25 ℃ of inducing culture 6-8 hour.Carry out result that 10%SDS-PAGE analyzes behind the cellular lysate as shown in Figure 3.As seen from the figure, expression of recombinant plasmid the differential protein of the about 70kD of molecular weight, conform to the molecular weight size of expectation B7-2-PE40KDEL.Record the 20-25% that this fusion rotein accounts for bacterial protein through scanning, see Fig. 4 for details.

Centrifugal gained thalline behind the inducing culture is suspended in the damping fluid of 20% sucrose, 30mmol/LTris-HCl (pH 7.4), 2mmol/L EDTA of new preparation ice bath 10 minutes.4 ℃ 1, centrifugal 10 minutes of 000g, cleer and peaceful precipitation in the collection, supernatant is outer pericentral siphon part, and 4 ℃ of storages are standby.Precipitation is resuspended in 50mmol/L Tris-HCl (pH.4), the 2mmol/L EDTA solution, ultrasonication, 4 ℃ 12,000 centrifugal 15 minutes, supernatant is mainly solubility expression part in the tenuigenin.The precipitation part comprises cell membrane component and insoluble inclusion body.Analyze by the external periplasm protein of polyacrylamide gel electrophoresis, cytoplasm protein and membranin, to determine the position of target protein at cell inner expression.Found that this fusion rotein almost all is that soluble protein exists.With the anti-people B7 monoclonal antibody of dilution in 1: 500 and anti-PEA polyclone is anti-and the rabbit anti-mouse igg effect of 1: 1000 alkali phosphatase enzyme mark after, develop the color with NBT/BCIP.This Western-blot result shows that expression product can resist with B7-2 monoclonal antibody and PE40 more, and specific reaction takes place, and the strong reaction band occurs at the 70kD place, and contrast bacterium corresponding position does not then have the immunoblotting band.The results are shown in Figure 5.After the 10%SDS-PAGE protein electrophoresis is carried out in sample preparation respectively with purified target protein liquid, transfer on the nitrocellulose filter.Sealed 2 hours with the calf serum/PBS/ sodium azide confining liquid that contains 10%, add anti-hatching 1～2 hour then; With PBS washing three times, with 0.1mmol/LTris-HCL, 0.05mol/L NaCL washing, phosphoric acid salt is cleaned again then as far as possible; Use 10% calf serum, Tris-HCL then, anti-as two antibody diluents two, two anti-ly hatched 1 hour 1 with film; At least wash three times with 0.1mol/l Tris-HCL, 0.15mol/lNaCL subsequently, develop the color with the NBT/BCIP test kit at last, to suitable colour developing degree distilled water flush away staining fluid termination reaction.

Embodiment 4: the purifying of recombination fusion protein B7-2-PE40KDEL

Method according to embodiment 3, the expansion scale, the a large amount of abduction deliverings of host cell BL21-CodonPlus (DE3)-RIL (Invitrogen company) that will comprise expression vector pRSETA-B7-2-PE40KDEL, with nutrient solution in centrifugal 20 minutes of 4 ℃ of 4500rpm, separated and collected is expressed and is gone up cleer and peaceful thalline, and expressing fusion protein content is about 25% in the mensuration supernatant.

For the solubility expression supernatant, under non-denatured state, utilize the Ni-NTA resin to carry out affinitive layer purification.With the balance liquid balance metal chelate chromatography post of 3 times of volumes, will be splined on Ni-NTA resin chromatography column through the filtering inclusion body supernatant of 0.45um, flow velocity is 1ml/min, and 4 ℃ of effect 60min flow out liquid and collection, and this is for passing peak (flow-through); Again after the balance liquid flushing with 3 times of volumes, with washings (50mmol/l NaH ₂PO ₄, 300mmol/l NaCl, the 40mmol/l imidazoles pH8.0) washes to ultraviolet absorption value and no longer changes; With elutriant (50mmol/lNaH ₂PO ₄, 300mmol/l NaCl, the 250mmol/l imidazoles pH8.0) is washed post and Fractional Collections; 10%SDS-PAGE is carried out in the various compositions samplings of collecting and Western Blot carries out Analysis and Identification, detect the result of purifying.

The sample that will contain target protein is fully dialysed 24 hours to remove imidazoles to dialyzate.The MonoQ chromatography column is with containing 10%B liquid (50mmol/L Na2HPO4,2mM EDTA, 1mol/L NaCl, pH7.2) A solution (50mmol/L Na2HPO4,2mM EDTA, pH7.2) after the balance, the sample that will contain target protein is with sample on the 1ml/min flow velocity, after containing the A liquid balance chromatography column of 10%B liquid with 3ml again, carry out linear gradient elution, promptly through 20 minutes B liquid hold-ups by 0 to 100%, flow velocity is 1ml/Min, collect each wash-out composition, Fractional Collections combines with collecting by the peak.10%SDS-PAGE and WesternBlot Analysis and Identification purified product.

Concentrate the liquid of anionresin purified target protein with the YM10 ultra-filtration membrane, be splined on the good Superdex of balance 75 gel columns, (50mmol/l Na2HPO4 (pH7.2), 2mmol/lEDTA, 0.2mol/l NaCl) separates with C liquid, flow velocity 0.3ml/min, collect target protein, Fractional Collections combines the every 1ml volume collection in place, no peak with collecting by the peak, play every 0.5ml volume and collect at the place, peak, and 10%SDS-PAGE detects the result of protein purification.

The mensuration of reorganization fusion toxin protein B 7-2-PE40KDEL protein content

Xylene Brilliant Cyanine G G-250 method (Bradford method): take by weighing Xylene Brilliant Cyanine G G-250 100mg and be dissolved in 95% ethanol of 50ml, add 100ml 85% phosphoric acid, adding distil water is settled to 1000ml, and the dye liquor that is made into 0.1mg/L is standby.Standard protein solution is prepared by BSA, in proportion the dilution standard protein solution.Add 100 μ l protein solutions in the 5ml dye liquor, shake up, placed 2-5 minute, 595nm wavelength place measures absorption value, makes typical curve.

The molecular weight analyse of reorganization B7-2-PE40KDEL fusion rotein

By 12%SDS-PAGE protein electrophoresis and gel imaging system analysis, carry out relative mobility and calculate, the relative molecular weight of the B7-2-PE40KDEL fusion rotein that obtains recombinating is 72.628kD, and 69.561kD compares with the theoretical prediction value, and error is within 5%.

Embodiment 5: the toxic preliminary evaluation of reorganization fusion toxin protein B 7-2-PE40KDEL selecting cell

Adopt mtt assay, the people T lymphoma cell line Jurkat of high expression level CD28 is suspended in the RPMI-1640 that contains 10% calf serum, the adjustment cell concn is 2 * 105/ml, add in 96 well culture plates, add the B7-2-PE40KDEL fusion rotein purification of samples of dilution by a certain percentage again, after hatching 48 hours under 37 ℃, 5%CO2 culture condition, add 10ul MTT (5mg/ml), continue to cultivate after 6 hours, the 570nm wavelength is measured the cytotoxic activity that the extinction density value calculates fusion rotein.Simultaneously and with the negative people B leukemic lymphoblastoid clone Raji that expresses of CD28 as negative control.Adopt trypan blue dyeing direct viewing cells survival state under light microscopic simultaneously.

The result of MTT shows, recombination fusion protein B7-2-PE40KDEL has significant lethal effect to the Jurkat cell in the 0.2ug/ml-2ug/ml concentration range, even the Jurkat cell just being had significant lethal effect being low to moderate under the dosage of 10ng/ml, dosage reaches its killing-efficiency of 1000ng/ml can reach 90%.The results are shown in Figure 6.Calculating its ID50 is 0.160ug/ml.

Adding fusion rotein after 48-72 hour, visible Jurkat nucleus shrinkage under the opticmicroscope, cell loses refractivity, and obviously increases with the increase dead cell of concentration, and becomes concentration dependent.Trypan blue exclusion result also proves, can kill and wound the Jurkat cell specifically at designed concentration range endonexin.And to the Raji cell of CD28 feminine gender, even if all do not observe any lethal effect when 2ug high dosage scope, the growth of Raji cell is as usual.The brand-new type recombinant immune of our designed structure of tentative confirmation suppresses fusion toxin protein B 7-2-PE40KDEL the CD28 positive target cell is had selective killing effect thus.

Owing to the static T cell expressing CD28 of 95% CD4+ and 50% CD8+, induced the expression of CTLA4 molecule after the activation again.Therefore, novel reorganization B7-PE40KDEL toxin protein fusion rotein of the present invention in vivo and in vitro can selective killing participates in the most CD4+ and the CD8+T cell of transplant rejection, reach the purpose of lasting control GVHD and HVGD and inducing specific immunological tolerance, make body still preserve certain immunizing power and anti-infection ability again.By reorganization B7-PE40KDEL toxin protein fusion rotein inductive immunological tolerance is to be difficult for reversing, the influence that not whether be harmonious by MHC, and xenogenesis organ or tissue will be transplanted becomes possibility.

Described herein and claimed invention is not limited in the scope of disclosed specific embodiments, because these embodiments are intended to illustrate several aspect of the present invention.The embodiment of any equivalence includes within the scope of the invention.In fact, except shown in this paper and described, after the description of reference front, be conspicuous to those skilled in that art for modification of the present invention.These modifications are also contained in the scope of appended claims.

Sequence table

＜110〉China People's Liberation Army Military Medical Sciences Academy Affiliated Hospital

＜120〉a kind of novel immunosuppression fusion rotein, its coding nucleic acid and uses thereof

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50 55 60

Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr

65 70 75 80

Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu

85 90 95

His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro

100 105 110

Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile

115 120 125

Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu

130 135 140

Asn Gly Glu Glu Leu Ser Ala Ile Asn Thr Thr Val Ser Gln Asp Pro

145 150 155 160

Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr

165 170 175

Thr Asn His Ser Phe Thr Cys Leu Ile Lys Tyr Gly His Leu Arg Val

180 185 190

Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp

195 200 205

Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

210 215 220

Gly Gly Gly Gly Ser Ser Gly Ser Leu Ala Ala Leu Thr Ala His Gln

225 230 235 240

Ala Cys His Leu Pro Leu Glu Thr Ser Thr Arg His Arg Gln Pro Arg

245 250 255

Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val

260 265 270

Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val

275 280 285

Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu

290 295 300

Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala

305 310 315 320

Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu

325 330 335

Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala

340 345 350

Ala Gly Glu Cys Val Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu

355 360 365

Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val

370 375 380

Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu

385 390 395 400

Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr

405 410 415

His Gly Thr Phe Leu Glu Ala Ser Gln Ser Ile Val Phe Gly Gly Val

420 425 430

Arg Ala Arg Asn Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile

435 440 445

Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro

450 455 460

Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val

465 470 475 480

Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala

485 490 495

Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu

500 505 510

Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg

515 520 525

Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile

530 535 540

Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp

545 550 555 560

Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp

565 570 575

Tyr Ala Ser Gln Pro Gly Lys Pro Pro Lys Asp Glu Leu

580 585

<210>5

<211>20

<212>DNA

＜213〉artificial sequence

<400>5

gcggatccgc tgctcctctg 20

<210>6

<211>24

<212>DNA

＜213〉artificial sequence

<400>6

cccgggttaa ggaatgtggt ctgg 24

<210>7

<211>8

<212>DNA

＜213〉artificial sequence

<400>7

gcggatcc 8

<210>8

<211>9

<212>DNA

＜213〉artificial sequence

<400>8

cccgggtta 9

<210>9

<211>678

<212>DNA

<213>

<400>9

atggctgctc ctctgaagat tcaagcttat ttcaatgaga ctgcaggcct gccgtgccaa 60

tttgcaaact ctcaaaacca aagcctgagt gagctagtag tattttggca ggaccaggaa 120

aacttggttc tgaatgaggt atacttaggc aaagagaaat ttgacagtgt tcattccaag 180

tatatgggcc gcacaagttt tgattcggac agttggaccc tgagacttca caatcttcag 240

atcaaggaca agggcttgta tcaatgtatc atccatcaca aaaagcccac aggaatgatt 300

cgcatccacc agatgaattc tgaactgtca gtgcttgcta acttcagtca acctgaaata 360

gtaccaattt ctaatataac agaaaatgtg tacataaatt tgacctgctc atctatacgc 420

ggttacccag aacctaagaa gatgagtgtt ttgctaagaa ccaagaattc aactatcgag 480

tatgatggta ttatgcagaa atctcaagat aatgtcacag aactgtacga cgtttccatc 540

agcttgtctg tttcattccc tgatgttgcg agcaatatga ccatcttctg tattctggaa 600

actgacaaga cgcggctttt atcttcacct ttctctatag agcttgagga ccctcagcct 660

cccccagacc acattcct 678

<210>10

<211>226

<212>PRT

<213>

<400>10

Met Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe Asn Glu Thr Ala Gly

1 5 10 15

Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln Ser Leu Ser Glu Leu

20 25 30

Val Val Phe Trp Gln Asp Gln Glu Asn Leu Val Leu Asn Glu Val Tyr

35 40 45

Leu Gly Lys Glu Lys Phe Asp Ser Val His Ser Lys Tyr Met Gly Arg

50 55 60

Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg Leu His Asn Leu Gln

65 70 75 80

Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile His His Lys Lys Pro

85 90 95

Thr Gly Met Ile Arg Ile His Gln Met Asn Ser Glu Leu Ser Val Leu

100 105 110

Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile Ser Asn Ile Thr Glu

115 120 125

Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile Arg Gly Tyr Pro Glu

130 135 140

Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys Asn Ser Thr Ile Glu

145 150 155 160

Tyr Asp Gly Ile Met Gln Lys Ser Gln Asp Asn Val Thr Glu Leu Tyr

165 170 175

Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pro Asp Val Ser Ser Asn

180 185 190

Met Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys Thr Arg Leu Leu Ser

195 200 205

Ser Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln Pro Pro Pro Asp His

210 215 220

Ile Pro

225

<210>11

<211>624

<212>DNA

<213>

<400>11

gttatccacg tgaccaagga agtgaaagaa gtagcaacgc tgtcctgtgg tcacaatgtt 60

tctgttgaag agccggcaca aactcgcatc tactggcaaa aggagaagaa aatggtgctg 120

actatgatgt ctggggacat gaatatatgg cccgagtaca agaaccggac catctttgat 180

attactaata acctctccat tgtgatcctg gctctgcgcc catctgacga gggcacatac 240

gagtgtgttg ttctgaagta tgaaaaagac gctttcaagc gggaacacct ggctgaagtg 300

acgttatcag tcaaagctga cttccctaca cctagtatat ctgactttga aattccaact 360

tctaatatta gaaggataat ttgctcaacc tctggaggtt ttccagagcc tcacctctcc 420

tggttggaaa atggagaaga attaagtgcc atcaacacaa cagtttccca agatcctgaa 480

actgagctct atgctgttag cagcaaactg gatttcaata tgacaaccaa ccacagcttc 540

acgtgtctca tcaagtatgg acatttaaga gtgaatcaga ccttcaactg gaatacaacc 600

aagcaagagc attttcctga taac 624

<210>12

<211>208

<212>PRT

<213>

<400>12

Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys

1 5 10 15

Gly His Asn Val Ser Val Glu Glu Pro Ala Gln Thr Arg Ile Tyr Trp

20 25 30

Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn

35 40 45

Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn

50 55 60

Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr

65 70 75 80

Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His

85 90 95

Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser

100 105 110

Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys

115 120 125

Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn

130 135 140

Gly Glu Glu Leu Ser Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu

145 150 155 160

Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr

165 170 175

Asn His Ser Phe Thr Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn

180 185 190

Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn

195 200 205

<210>13

<211>22

<212>DNA

＜213〉artificial sequence

<400>13

gctgctcctc tgaagattca ag 22

<210>14

<211>73

<212>DNA

＜213〉artificial sequence

<400>14

cgacccacca ccgcccgagc caccgccacc gcttccaccg cctccagatc cgccgccacc 60

aggaatgtgg tct 73

<210>15

<211>27

<212>DNA

＜213〉artificial sequence

<400>15

cgcgaattca tggttatcca cgtgacc 27

<210>16

<211>82

<212>DNA

＜213〉artificial sequence

<400>16

cgacccacca ccgcccgagc caccgccacc gcttccaccg cctccagatc cgccgccacc 60

gttatcagga aaatgctctt gc 82

<210>17

<211>40

<212>DNA

＜213〉artificial sequence

<400>17

tcgggcggtg gtgggtcggg cggcagcctg gccgcgctga 40

<210>18

<211>40

<212>DNA

＜213〉artificial sequence

<400>18

tcccccgggg gattacttca ggtcctcgcg cggcggtttg 40

<210>19

<211>48

<212>DNA

＜213〉artificial sequence

<400>19

tcccccgggg gattacagct cgtccttcgg cggtttgccg ggctggct 48

Claims (24)

1. fusion rotein wherein comprises the syzygy that immune aglucon B7 or its can merge mutually with corresponding TXi Baoshouti bonded variant, derivative, analogue or fragment and toxin protein.

2. according to the fusion rotein of claim 1, wherein said immune aglucon is B7-1.

3. according to the fusion rotein of claim 1, wherein said immune aglucon is B7-2.

4. according to the fusion rotein of claim 1, wherein comprise born of the same parents' outside part of described immune aglucon.

5. according to the fusion rotein of claim 1, wherein said immune aglucon is the people.

6. according to the fusion rotein of claim 1, wherein said toxin protein is plant poison or bacteriotoxin.

7. according to the fusion rotein of claim 6, wherein said toxin protein is Ricin, phytohemagglutinin, diphtheria toxin, shiga toxin, Pseudomonas exotoxin or Pseudomonas aeruginosa extracellular toxin.

8. according to the fusion rotein of claim 1, it has aminoacid sequence shown in SEQ ID NO:2 or 4.

9. isolating polynucleotide, each fusion rotein among its coding claim 1-8.

10. nucleic acid construct, it comprises polynucleotide of claim 9 and the expression control sequenc that can be operatively connected with it.

11. an expression vector wherein comprises the nucleic acid construct of claim 10.

12. according to the expression vector of claim 11, it is pRSETA-B7-2-PE40KDEL.

13. comprise the host cell of the expression vector of the nucleic acid construct of claim 10 or claim 11 or 12.

14. according to the host cell of claim 13, it is protokaryon or eukaryotic cell.

15. according to the host cell of claim 14, it is a bacterium.

16. according to the host cell of claim 15, it is intestinal bacteria.

17. according to the host cell of claim 16, it is BL21-CodonpPlus (DE3)-RIL or BL21 (DE3) pLysS that comprises the pRSETA-B7-2-PE40KDEL recombinant expression vector.

18. a method for preparing each fusion rotein among the claim 1-8 comprises that (i) is being suitable under the condition of described expressing fusion protein cultivating each host cell of claim 13-17; (ii) from culture, reclaim described fusion rotein.

19. each fusion rotein can be used for suppressing purposes in over-drastic or the unnecessary immunoreactive medicine in preparation among the claim 1-8.

20. according to the purposes of claim 19, wherein said over-drastic or unnecessary immune response are that graft versus host repels or host versus graft is repelled, or autoimmune disorder.

21. according to the purposes of claim 20, wherein said autoimmune disorder is rheumatoid arthritis, lupus erythematosus, psoriasis, ankylosing spondylitis, myasthenia gravis, multiple sclerosis or insulin-dependent diabetes.

22. the polynucleotide of claim 10 can be used for suppressing purposes in the medicine of autoimmune disorder in preparation.

23. according to the purposes of claim 22, wherein said autoimmune disorder is rheumatoid arthritis, lupus erythematosus, psoriasis, ankylosing spondylitis, myasthenia gravis, multiple sclerosis or insulin-dependent diabetes.

24. a pharmaceutical composition, wherein contain the immunosuppression significant quantity claim 1-8 each fusion rotein or the polynucleotide of claim 9, and pharmaceutically acceptable carrier.

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