CN108727488A - The preparation and application of anti-norovirus GII.17 monoclonal antibodies - Google Patents

Abstract

The present invention provides the preparations and application of anti-norovirus GII.17 monoclonal antibodies, specifically mouse is immunized using the norovirus GII.17 virus-like particles of recombinant expression in the present invention, and the monoclonal antibody for norovirus GII.17 is prepared for by hybridoma technology.Through screening, obtained one plant can in conjunction with GII.17 monoclonal antibody, can not only specific recognition GII.17 virus-like particles, and have powerful neutralization activity.

Description

The preparation and application of anti-norovirus GII.17 monoclonal antibodies

Technical field

The invention belongs to biomedicine fields, specifically, the present invention relates to anti-norovirus GII.17 monoclonal antibodies Preparation and application.

Background technology

Norovirus (NoVs) is one of the main pathogen for leading to the Sporadic cases of acute gastroenteritis and breaking out greatly, and And norovirus can infect the people of all age brackets.Although caused symptom is generally relatively milder after norovirus infection, There is self-limited course to continue 1-3 days or so, but can still cause in the people of child, old man and immunologic inadequacy more tight The symptom of weight, or even cause death.According to the amino acid sequence of VP1 capsid proteins, norovirus can be divided into 6 genotype (G1-GVI) and multiple gene type, but only GI, GII and GIV can infect the mankind.The infection of people's norovirus mainly by promise such as Caused by viral GII, and big outbreak of epidemic is then caused by norovirus GII.4.But promise such as GII.17 Strain exists recently Different Asian countries causes breaking out for big acute gastroenteritis, and has norovirus in South America, North America and Europe The detection of GII.17.According to the sequence of VP1, newfashioned GII.17 Strain belongs to GII.17cluster C, in Asia Prevalence is simultaneously spread to rapidly all over the world, and GII.17, which may substitute GII.4 and be used as, in the near future leads to acute gastroenteritis Norovirus main pathogens.

Norovirus has prevalence in developed country and developing country, brings serious economic loss to various countries, gives Children, old man health cause prodigious threat.Up to the present, the preventative vaccine that does not list and special efficacy it is therapeutic Drug.Norovirus lacks simple cell culture model, and also without small animal model, this gives the research of vaccine and antiviral drugs Bring prodigious obstruction.Humanization mouse resource monoclonal antibody, which is an exploitation prevention and treatment virus infective medicament, efficacious prescriptions Method, while monoclonal antibody can also be used for the diagnosis of virus infection.The monoclonal antibody for GII.17 is not yet had been reported that at present.

Invention content

The purpose of the present invention is to provide a kind of preparations and application of anti-norovirus GII.17 monoclonal antibodies.

In the first aspect of the present invention, provide a kind of heavy chain variable region of antibody, the heavy chain variable region have with Under one or more complementary determining region CDR：

CDR1 shown in SEQ ID NO.1,

CDR2 shown in SEQ ID NO.2, and

CDR3 shown in SEQ ID NO.3.

In another preferred example, the heavy chain variable region has amino acid sequence shown in SEQ ID NO.4.

The second aspect of the present invention, provides a kind of heavy chain of antibody, and the heavy chain has first aspect present invention institute The heavy chain variable region and heavy chain constant region stated.

In another preferred example, the heavy chain constant region behaviour source or mouse source.

In another preferred example, the amino of the heavy chain of the antibody selects sequence as shown in SEQ ID NO.10.

The third aspect of the present invention provides a kind of light chain variable region of antibody, and the light chain variable region, which has, to be selected from down The complementary determining region CDR of group：

CDR1 ' shown in SEQ ID NO.5,

CDR2 ' shown in SEQ ID NO.6, and

CDR3 ' shown in SEQ ID NO.7.

In another preferred example, the light chain variable region has amino acid sequence shown in SEQ ID NO.8.

The fourth aspect of the present invention, provides a kind of light chain of antibody, and the light chain has third aspect present invention institute The light chain variable region and constant region of light chain stated.

In another preferred example, the constant region behaviour source or mouse source of the light chain.

The fifth aspect of the present invention, provides a kind of antibody, and the antibody has：

(1) heavy chain variable region as described in the first aspect of the invention；And/or

(2) light chain variable region as described in third aspect present invention.

In another preferred example, the antibody has：Heavy chain as described in respect of the second aspect of the invention；And/or the present invention the Light chain described in four aspects.

In another preferred example, the antibody is the antibody of the anti-norovirus GII.17 of specificity.

In another preferred example, the antibody includes：Single-chain antibody (scFv), monoclonal antibody, is fitted into double-chain antibody Antibody (such as human mouse chimeric antibody), mouse source antibody or humanized antibody.

The sixth aspect of the present invention, provides a kind of recombinant protein, and the recombinant protein has：

(i) sequence of heavy chain variable region as described in the first aspect of the invention, heavy chain as described in respect of the second aspect of the invention Sequence, the sequence of light chain variable region as described in third aspect present invention, the sequence of light chain as described in fourth aspect present invention The sequence of row or the antibody as described in fifth aspect present invention；

(ii) polypeptide, protein drug sequence；And

(iii) sequence label of optional assistance expression and/or purifying.

In another preferred example, the polypeptide protein drug be single-chain antibody (scFv), double-chain antibody, monoclonal antibody, Or chimeric antibody.

In another preferred example, the sequence label is selected from the group：6 × His labels, GGGS sequences, FLAG labels.

In another preferred example, the recombinant protein includes bispecific antibody, chimeric antibody.

The seventh aspect of the present invention provides a kind of polynucleotides, it encodes polypeptide selected from the group below：

(1) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair Light chain variable region described in the bright third aspect, the light chain as described in fourth aspect present invention, as described in fifth aspect present invention Antibody；Or

(2) recombinant protein as described in sixth aspect present invention.

In another preferred example, the polynucleotides have the institute of SEQ ID NO.13,14,15,16,17,18,11 or 9 The sequence shown.

The eighth aspect of the present invention provides a kind of carrier, it contains the polynucleotides described in seventh aspect present invention.

In another preferred example, the carrier includes：Bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, Mammalian cell virus such as adenovirus, retrovirus or other carriers.

The ninth aspect of the present invention provides a kind of genetically engineered host cell, it contains eighth aspect present invention The polynucleotides described in seventh aspect present invention are integrated in the carrier or genome.

The tenth aspect of the present invention provides a kind of immune conjugate, which contains：

(a) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair Light chain variable region described in the bright third aspect, the light chain as described in fourth aspect present invention, as described in fifth aspect present invention Antibody or the recombinant protein as described in sixth aspect present invention；With

(b) coupling moiety selected from the group below：Detectable marker, drug, toxin, cell factor, radionuclide or Enzyme.

In another preferred example, the conjugate is selected from：(magnetic is total by fluorescence or luminous marker, radioactively labelled substance, MRI Shake imaging) or CT (x-ray tomography of electronic computer) contrast agent or the enzyme of detectable product, radiation can be generated Property nucleic, biotoxin, cell factor (such as IL-2), antibody, antibody Fc fragment, antibody scFv fragment, gold nano grain/receive Rice stick, liposome, magnetic nanosphere or any type of nano particle etc..

The eleventh aspect of the present invention, provides a kind of pharmaceutical composition, it contains：

(i) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair Light chain variable region described in the bright third aspect, the light chain as described in fourth aspect present invention, as described in fifth aspect present invention Antibody, the recombinant protein as described in sixth aspect present invention or the immune conjugate as described in tenth aspect present invention；And

(ii) pharmaceutically acceptable carrier.

In another preferred example, the pharmaceutical composition is injection type.

The twelveth aspect of the present invention provides heavy chain variable region as described in the first aspect of the invention, such as present invention the Two aspect described in heavy chain, the light chain variable region as described in third aspect present invention, the light chain as described in fourth aspect present invention, Antibody as described in fifth aspect present invention, the recombinant protein as described in sixth aspect present invention or such as tenth aspect present invention The purposes of the immune conjugate is used to prepare medicament, reagent, detection plate or kit.

In another preferred example, the reagent includes the immune particle of chip, coated antibody.

The thirteenth aspect of the present invention, provides a kind of preparation method of recombinant polypeptide, and this method includes：

(a) under conditions suitable for the expression, the host cell described in the 9th face of the invention is cultivated；

(b) recombinant polypeptide is isolated from culture, the recombinant polypeptide is the antibody described in fifth aspect present invention Or the recombinant protein described in sixth aspect present invention.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Description of the drawings

The anti-GII.17 monoclonal antibodies of Fig. 1 Polyacrylamide Gel Electrophoresis purifying.The antibody of 5 kinds of purifying is respectively through containing also It is loaded in 12% polyacrylamide gel after the buffer solution processing of former agent and carries out electrophoresis, and shown with coomassie brilliant blue staining Protein band.M, Protein Marker；1,1D3-2 monoclonal antibody；2,3A3-3 monoclonal antibodies；3,2C1-1 monoclonal antibodies；4,4B1-2 monoclonal antibodies.

Fig. 2 enzyme-linked immunosorbent assay (Elisa) identifies the binding ability of monoclonal antibody and not synantigen.It is every on Elisa plates Hole is coated with 50ngGII.7 (Fig. 2A), GII.4 (Fig. 2 B) or GI.1 (Fig. 2 C) virus-like particle respectively, adds difference respectively per hole The monoclonal antibody of the purifying of concentration is incubated 2 hours at 37 DEG C, is then incubated with the HRP anti-mouse secondary antibodies marked.Anti-hepatitis B surface is anti- Former (HBsAg) monoclonal antibody is used to do unrelated control.Each point shows the OD450nm average values that three repeat samples measure in figure And standard deviation.

Fig. 3 .Western blot analyses.Virus-like particle after processing, on in 12% polyacrylamide gel into Row electrophoresis, is then transferred on pvdf membrane, is hybridized with the monoclonal antibody of purifying.1, GI.1 virus-like particle；2, GII.3 virus-likes Particle；3, GII.17 virus-like particles；Control, anti-hepatitis B surface antigen (HBsAg) monoclonal antibody.

The sandwich Elisa of Fig. 4 detect GII.17 virus-like particles.It is coated with 50ul1 respectively per hole on Elisa plates:2000 is dilute The rabbit-anti GII.17 serum released adds the GII.17 virus-like particles of various concentration to be incubated 2 hours at 37 DEG C, then often respectively per hole The monoclonal antibody of the purifying of 50ng is added in hole, is finally incubated with the HRP anti-mouse secondary antibodies marked.Anti-hepatitis B surface antigen (HBsAg) Monoclonal antibody is used to do unrelated control.

The identification of the monoclonal antibody of Fig. 5 DNA recombinant expressions.It is coated with 50ngGII.4 diseases respectively per hole on Elisa plates Malicious sample particle adds the monoclonal antibody of the purifying of various concentration to be incubated at 37 DEG C 2 hours, the anti-mouse two then marked with HRP respectively per hole It is anti-to be incubated.The culture supernatant of the cell of untransfected plasmid is as blank control.Block diagram shows three repeating samples in figure The OD450nm average values and standard deviation that product measure.

Specific implementation mode

The present inventor's in-depth study by extensive by, being prepared for using GII.17 virus-like particles as immunogene can be special Property identification GII.17 monoclonal antibody.Elisa and replacement neutralize the methods of experiment and illustrate that these antibody can be used for sensitive inspection Survey and analyze GII.17, it is often more important that some monoclonal antibodies also have powerful neutralization activity.

Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experiment conditions, because this Class method and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and And it is not intended to be restrictive, the scope of the present invention will be limited only by the claims which follow.

Unless otherwise defined, otherwise whole technologies used herein all have with scientific terminology such as fields of the present invention The normally understood identical meanings of those of ordinary skill.As used herein, in use, term in mentioning the numerical value specifically enumerated " about " mean that the value can change not more than 1% from the value enumerated.For example, as used herein, statement " about 100 " includes 99 Hes 101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).

Although can be used and heretofore described similar or of equal value any method in the implementation or test of the present invention And material, herein place enumerate preferred method and material.

Norovirus

Norovirus is one of the main pathogens for leading to children and adult acute's gastroenteritis.Epidemic data shows, Norovirus GII.4 is to cause the main pathogens of norovirus outbreak of epidemic, but norovirus GII.17 is in Asia recently Various countries cause a large amount of acute gastroenteritis Breakout events, and have norovirus GII.17's in South America, North America and Europe Detection.This brings serious economic loss to various countries, also causes prodigious threat to the health of children, old man.To current Until, it is not directed to the preventative vaccine and curative drug of norovirus GII.17.The present invention utilizes the promise of recombinant expression such as Mouse is immunized in viral GII.17 virus-like particles, is prepared for resisting for the monoclonal of norovirus GII.17 by hybridoma technology Body.Through screening, obtained four plants can in conjunction with GII.17 monoclonal antibodies, be respectively designated as 1D3-2,2C1-1,3A3-3 and 4B1-2.ELISA and Western blot experimental results show that 1D3-2,2C1-1,3A3-3 and 4B1-2 can specific recognitions GII.17 virus-like particles, lowest detection limit are respectively 0.078ng, 0.078ng, 0.156ng and 0.156ng.In addition, substituting It neutralizes experiment and shows that monoclonal antibody 1D3-2 and 3A3-3 are provided with powerful potential neutralization activity.In conclusion these antibody are not only Test in laboratory tool, and be the useful examination of the reliable candidate and exploitation diagnostic method that prepare therapeutic humanization monoclonal antibody Agent.

Antibody

As used herein, term " antibody " or " immunoglobulin " are about 150000 dalton for having identical structure feature Different four glycan albumen is made of two identical light chains (L) and two identical heavy chains (H).Every light chain is total by one Valence disulfide bond is connected with heavy chain, and the disulfide bond number between the heavy chain of different Immunoglobulin Isotypes is different.Each heavy chain and The intrachain disulfide bond at light chain also regular interval.There is variable region (VH) in one end of each heavy chain, is followed by multiple constant regions.Every There is variable region (VL) in one end of light chain, and the other end has constant region；The constant region of light chain is opposite with the first of heavy chain constant region, gently The variable region of chain is opposite with the variable region of heavy chain.Special amino acid residue forms boundary between light chain and the variable region of heavy chain Face.

As used herein, term is " variable " indicates that certain parts of variable region in antibody are different in sequence, its shape The combination to its specific antigen and specificity at various specific antibodies.However, changeability and being unevenly distributed over entire anti- In body variable region.It concentrates in light chain and heavy chain variable region three segments being known as in complementary determining region (CDR) or hypervariable region In.More conservative part is known as framework region (FR) in variable region.Four FR are respectively contained in the variable region of native heavy and light chain Area, they are in generally beta sheet configuration, are connected by three CDR of formation connection ring, can form part β foldings in some cases Stack structure.CDR in every chain is by the areas FR firmly against the antigen for together forming antibody together and with the CDR of another chain Binding site (referring to Kabat etc., NIH Publ.No.91-3242, rolls up I, 647-669 pages (1991)).Constant region is not joined directly With the combination of antibody and antigen, but they show different effector functions, such as participate in antibody dependent on the thin of antibody Cellular toxicity.

As it is known by the man skilled in the art, immune conjugate and fusion expressed product include：Drug, toxin, cell factor (cytokine), radionuclide, enzyme and other diagnosis or treatment molecule and antibody of the invention or its segment in conjunction with and formed Conjugate.The invention also includes the cell surface marker objects combined with the anti-norovirus GII.17 antibody or its segment Or antigen.

As used herein, term " heavy chain variable region " and " V_H" be used interchangeably.

As used herein, term " variable region " and " complementary determining region (complementarity determining Region, CDR) " it is used interchangeably.

In the preferred embodiment of the present invention, the heavy chain variable region of the antibody includes that three complementations are determined Determine area CDR1, CDR2 and CDR3, wherein

CDR1：GYTFSSYW, SEQ ID NO.1；

CDR2：ILPGNDNS, SEQ ID NO.2；

CDR3：ARSTWDKGYYYPLDY, SEQ ID NO.3.

In another preferred example, the heavy chain variable region has amino acid sequence shown in SEQ ID NO.4：

QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKLRPGHGLEWIGEILPGNDNSNYNKKFKGKATFTADTSSN TAYIQLGSLTSEDSAVYYCARSTWDKGYYYPLDYWGQGTSVTV, SEQ ID NO.4.

In the preferred embodiment of the present invention, the heavy chain of the antibody includes above-mentioned heavy chain variable region and heavy chain Constant region.

As used herein, term " light chain variable region " and " V_L" be used interchangeably.

In the preferred embodiment of the present invention, the light chain variable region of the antibody includes three complementary determining regions CDR1 ', CDR2 ' and CDR3 ', wherein

CDR1'：SSINY, SEQ ID NO.5；

CDR2'：DTS, SEQ ID NO.6；

CDR3'：HQRSSSPWT, SEQ ID NO.7.

In another preferred example, the heavy chain variable region has amino acid sequence shown in SEQ ID NO.8：

QIVLTQSPAIMSASPGEKVTLTCSASSSINYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSM EAEDAATYHCHQRSSSPWTFGGGTELEIK, SEQ ID NO.8.

In the preferred embodiment of the present invention, the heavy chain of the antibody includes above-mentioned heavy chain variable region and heavy chain Constant region.

In the present invention, term " antibody of the present invention ", " albumen of the present invention " or " polypeptide of the present invention " is used interchangeably, all Refer to the polypeptide of specific binding norovirus GII.17, such as the albumen with above-mentioned heavy chain variable region and/or light chain variable region Or polypeptide.They can be with or without initial methionine.

The present invention also provides other protein or fusion expressed product with antibody of the present invention.Specifically, of the invention Include with the heavy chain containing variable region any protein or protein conjugate and fusion expressed product (i.e. immune conjugate and Fusion expressed product), as long as the variable region is identical as the heavy chain variable region of antibody of the present invention or at least 90% homology, preferably At least 95% homology.

Generally, the antigenic binding property of antibody can referred to as may be used by being described positioned at 3 specific regions of heavy chain variable region Become region (CDR), by this it is intersegmental be divided into 4 frame areas (FR), the amino acid sequence of 4 FR is relatively conservative, not directly Participate in association reaction.These CDR form cyclic structure, and the β-pleated sheet formed by FR therebetween is close to each other on space structure, The CDR on CDR and corresponding light chain on heavy chain constitutes the antigen binding site of antibody.It can be by comparing the antibody of same type Amino acid sequence determine be which Amino acid profile FR or CDR region domain.

The heavy chain of antibody of the present invention and/or the variable region of light chain are particularly interesting, because at least partly being related in them And combine antigen.Therefore, the present invention, which includes those, has the heavy chain of antibody with CDR and/or the molecule of light chain variable region, as long as its The CDR and CDR identified herein has the homology of 90% or more (preferably 95% or more, most preferably 98% or more).

The present invention includes not only complete antibody, further includes the segment or antibody and other sequences with immunocompetent antibody Arrange the fusion protein formed.Therefore, the invention also includes the segment of the antibody, derivative and analogue.

As used herein, term " segment ", " derivative " and " analog " refer to that be kept substantially antibody of the present invention identical Biological function or active polypeptide.Polypeptide fragment, the derivative or the like of the present invention can be (i) there are one or it is multiple Conservative or non-conservative amino acid residue (preferably conservative amino acid) substituted polypeptide, and such substituted amino Sour residue can may not be by genetic code encoding, or (ii) has substitution in one or more amino acid residues The polypeptide of group, or (iii) mature polypeptide and another compound (for example extend the compound of polypeptide half-life period, such as poly- second Glycol) fusion is formed by polypeptide, or (iv) additional amino acid sequence is fused to this polypeptide sequence and the polypeptide that is formed is (as before Lead sequence or secretion sequence or for purifying the sequence or proprotein sequence of this polypeptide, or egg is merged with what 6His labels were formed In vain).According to the teaching of this article, these segments, derivative and analogue belong to scope known to those skilled in the art.

Antibody of the present invention refers to the polypeptide that active including above-mentioned CDR region is combined with norovirus GII.17.The term is also Include with antibody identical function of the present invention, polypeptide comprising above-mentioned CDR region variant form.These variant forms include (but being not limited to)：One or more (being usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) Missing, insertion and/or the substitution of amino acid, and C-terminal and/or N-terminal addition it is one or several (be usually 20 with It is interior, be more preferably within 5 within preferably 10) amino acid.For example, in the art, use is similar in performance When amino acid is replaced, the function of protein is not usually changed.For another example, C-terminal and/or N-terminal add one or Several amino acid will not generally also change the function of protein.The term further includes that the active fragment of antibody of the present invention and activity are spread out Biology.

The variant form of the polypeptide includes：Homologous sequence, conservative variant, allelic variant, natural mutation, induction Mutant, the encoded albumen of DNA that can hybridize with the coding DNA of antibody of the present invention under the conditions of high or low stringency, with And more peptide or proteins of the antiserum acquisition using anti-antibody of the present invention.

The present invention also provides other polypeptides, such as the fusion protein comprising human antibody or its segment.In addition to almost overall length Outside polypeptide, the invention also includes the segments of antibody of the present invention.In general, the segment has at least about 50 companies of antibody of the present invention Continue amino acid, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, most preferably at least about 100 continuous amino acids.

In the present invention, " conservative variant of antibody of the present invention " refers to compared with the amino acid sequence of antibody of the present invention, There are at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid are with similar or analogous properties Amino acid is replaced and forms polypeptide.These conservative variation's polypeptides carry out amino acid substitution preferably based on Table I and generate.

Table I

Initial residue	Representative substitution	Preferred substitution
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The present invention also provides encoding such antibodies or the polynucleotide molecules of its segment or its fusion protein.The present invention's Polynucleotides can be DNA form or rna form.DNA form includes cDNA, genomic DNA or artificial synthesized DNA.DNA can To be single-stranded or double-strand.DNA can be coding strand or noncoding strand.

The polynucleotides of mature polypeptide for encoding the present invention include：The coded sequence of encoding mature polypeptide；Mature polypeptide Coded sequence and various additional coding sequences；The coded sequence (and optional additional coding sequence) of mature polypeptide and non-volume Code sequence.

Term " polynucleotides of coding polypeptide " can be the polynucleotides for including this polypeptide of coding, can also be to further include The polynucleotides of additional code and/or non-coding sequence.

The invention further relates to hybridizing with above-mentioned sequence and having at least 50% between two sequences, preferably at least 70%, more preferably at least polynucleotides of the 80% phase same sex.The present invention is more particularly directed under strict conditions with it is of the present invention more The interfertile polynucleotides of nucleotide.In the present invention, " stringent condition " refers to：(1) compared with low ionic strength and higher temperature Under hybridization and elution, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant, such as 50% (v/v) formyl when (2) hybridization Amine, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or the phase same sex of (3) only between two sequences at least 90% with On, more preferably 95% or more when, just hybridizes.Further, the polypeptide of interfertile polynucleotide encoding and SEQ ID NO.:32- Mature polypeptide shown in one of 37 has identical biological function and activity.

The nucleotide full length sequence or its segment of the antibody of the present invention can usually use PCR amplification method, recombination method or artificial Synthetic method obtains.A kind of feasible method is that manually synthetic method synthesizes related sequence, especially fragment length When shorter.In general, by first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.In addition, may be used also The coded sequence of heavy chain and expression label (such as 6His) are merged, fusion protein is formed.

Once obtaining related sequence, so that it may to obtain related sequence in large quantity with recombination method.This is typically will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method. Biomolecule (nucleic acid, albumen etc.) according to the present invention includes existing biomolecule in a separate form.

At present, it is already possible to completely by chemical synthesis come obtain encoding albumen of the present invention (its segment or its derivative Object) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or such as carrier) and In cell.In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.

The invention further relates to the carriers for including above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence.This A little carriers can be used for converting host cell appropriate, allow it to expression protein.

Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high Equal eukaryocytes, such as mammalian cell.Representative example has：Escherichia coli, streptomyces；Salmonella typhimurium it is thin Bacterium cell；Fungal cell's such as yeast；The insect cell of drosophila S2 or Sf9；The zooblast etc. of CHO, COS7,293 cells.

It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original When core biology such as Escherichia coli, can absorb the competent cell of DNA can harvest after exponential phase of growth, use CaCl₂Method processing, institute With the step of it is generally well-known in the art.Another method is to use MgCl₂.If desired, conversion can also use the side of electroporation Method carries out.When host is eucaryote, following DNA transfection methods can be selected：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

The transformant of acquisition can use conventional method culture, express the polypeptide of the coded by said gene of the present invention.According to used Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of suitable for host cell growth It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction) Cell is further cultured for a period of time by the promoter for inducing selection.

Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as Fruit needs, its physics, chemical and other characteristics can be utilized to be separated by various separation methods and purify the albumen of recombination.This A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to：The renaturation process of routine is used Protein precipitant handles (salting-out method), centrifugation, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.

The antibody of the present invention can be used alone, also can be with detectable marker (for diagnostic purpose), therapeutic agent, PK (eggs White kinases) combination of modified part or any the above substance combines or coupling.

Detectable marker for diagnostic purposes includes but not limited to：Fluorescence or luminous marker, radioactively labelled substance, MRI (magnetic resonance imaging) or CT (x-ray tomography of electronic computer) contrast agent can generate detectable product Enzyme.

The therapeutic agent that can be combined or be coupled with antibody of the present invention includes but not limited to：1. radionuclide (Koppe etc., 2005, (Cancer metastasis reviews) 24,539 is commented in metastasis of cancer)；2. biology poison (Chaudhary etc., 1989, Natural (Nature) 339,394；Epel etc., 2002, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 51,565)；3. cell factor such as IL-2 etc. (Gillies etc., 1992, National Academy of Sciences proceeding (PNAS) 89,1428；Card etc., 2004, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 53,345；Halin etc., 2003, cancer research (Cancer Research) 63,3202)；4. gold nano (Lapotko etc., 2005, cancer communicates (Cancer letters) 239,36 to particle/nanometer rods；Huang etc., 2006, the U.S. Chemical Society's magazine (Journal of the American Chemical Society) 128,2115)；5. virion (Peng etc., 2004, gene therapy (Gene therapy) 11,1234)；6. liposome (Mamot etc., 2005, cancer research (Cancer research) 65,11631)；7. magnetic nanosphere；8. pro-drug activation enzymes are (for example, DT- diaphorases (DTD) or connection Phenyl hydrolase-sample protein (BPHL))；10. chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..

The present invention also provides a kind of compositions.In preference, the composition is pharmaceutical composition, it contains The antibody stated or its active fragment or its fusion protein and pharmaceutically acceptable carrier.In general, these substances can be prepared In nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein pH ordinarily is about 5-8, and preferably pH is about 6-8, although pH value can be varied from the property and illness to be treated that are formulated substance.Prepared pharmaceutical composition It can be administered by conventional route, including (but being not limited to)：Intravenously or local administration.

The pharmaceutical composition of the present invention can be directly used for combining norovirus GII.17 molecules, thus can be used for preventing and control Treat norovirus infection.In addition, also can be used simultaneously other therapeutic agents.

The pharmaceutical composition of the present invention contain safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more Good ground 0.1-80wt%) the above-mentioned antibody (or its conjugate) and pharmaceutically acceptable carrier or excipient of the present invention. This kind of carrier includes (but being not limited to)：Brine, buffer solution, glucose, water, glycerine, ethyl alcohol, and combinations thereof.Pharmaceutical preparation is answered Match with administering mode.The pharmaceutical composition of the present invention can be made into injection form, such as with physiological saline or contain Portugal The aqueous solution of grape sugar and other adjuvants is prepared by conventional method.Pharmaceutical composition such as injection, solution are preferably in aseptic condition Lower manufacture.The dosage of active constituent is therapeutically effective amount, such as about 5 mg/kg body of about 1 microgram/kg body weight-daily Weight.In addition, the polypeptide of the present invention can be also used together with other therapeutic agents.

It is that the immune conjugate of safe and effective amount is applied to mammal when using pharmaceutical composition, the wherein safety Effective quantity typically at least about 10 micrograms/kg body weight, and 8 mg/kg weight are in most cases no more than about, preferably The ground dosage is about 1 mg/kg weight of about 10 micrograms/kg body weight-.Certainly, specific dosage is also contemplated that administration route, disease The factors such as people's health status, within the scope of these are all skilled practitioners technical ability.

The immunoglobulin of label

In the preference of the present invention, the antibody carries detectable marker.More preferably, marker choosing From the following group：Colloid gold label object, colored labels or fluorescent marker.

Method known to those skilled in the art progress can be used in colloid gold label.In the preferred side of the present invention In case, the monoclonal antibody colloid gold label of norovirus GII.17 obtains the monoclonal antibody of colloid gold label.

The norovirus GII.17 monoclonal antibodies of the present invention have specificity well, very high potency.

Method and sample

The present invention relates in the method for cell and/or the pattern detection norovirus GII.17 of histolysis.It should Method and step approximately as：Obtain cell and/or tissue samples；In the medium by sample dissolving；Detect the sample in the dissolving The level of norovirus GII.17 in this.Sample used in the method for the present invention can be present in cell-preservation liquid to include Any sample of cell, as used in liquid basal cell detection method.

Kit

The present invention also provides a kind of reagents for the detection plate referring to the antibody (or its segment) containing the present invention or the present invention Box, in the preference of the present invention, the kit further includes container, operation instructions, buffer etc..

The present invention is further designed for the detection kit of detection norovirus GII.17 levels, which includes knowing The antibody of other norovirus GII.17, the cracking medium for dissolving sample detect required common reagent and buffer solution, such as each Kind buffer solution, detection label, detection substrate etc..The detection kit can be in-vitro diagnosis device.

Main advantages of the present invention are：

(1) a kind of anti-norovirus GII.17 monoclonal antibodies are provided for the first time；

(2) anti-norovirus GII.17 monoclonal antibodies provided by the invention can not only the delicately non-change of specific recognition Property norovirus GII.17 sample particles, and with combine denaturation norovirus GII.17 virus-like particles ability.

(3) anti-norovirus GII.17 monoclonal antibodies provided by the invention have powerful potential neutralization activity.

(4) anti-norovirus GII.17 monoclonal antibodies provided by the invention can identify the norovirus GI.1 after denaturation Virus-like particle and norovirus GII.4 virus-like particles.

With reference to specific embodiment, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate this hair It is bright rather than limit the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to routine The works such as condition such as U.S. Sambrook.J《Molecular Cloning: A Laboratory room guide》(Huang Peitang etc. is translated, Beijing：Science Press, 2002 Year) described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number press weight Amount calculates.Experiment material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.

Material and method

1. antigen prepares and mouse immune

Using pichia yeast expression system, virus-like particle is prepared for by expressing norovirus GII.17VP1.By 5ug Virus-like particle (50ul volumes) mixed with isometric aluminium adjuvant (500ug) pneumoretroperitoneum be immunized 6 weeks female Balb/c mouse, 0 week, 2 weeks, 4 weeks it is each immune primary.At the 6th week, take mice serum detection for GII.17 virus-like particle specificity The titre of antibody and the effect for inhibiting GII.17 virus-like particles to be combined with porcine gastric mucin III.At the 7th week, specificity is chosen Antibody titer highest and the preferable mouse of the effect for inhibiting GII.17 virus-like particles to be combined with porcine gastric mucin III are logical Cross tail vein booster immunization 15ug GII.17 virus-like particles.After 3 days, mouse spleen is taken to be used to prepare hybridoma.

2. the preparation and screening of hybridoma cell strain

After mouse tail vein booster immunization 3 days, Mouse spleen cells is taken to melt by PEG1500 with myeloma cell SP2/0 It closes, prepares hybridoma.After 9 days, specific secretion is screened by enzyme-linked immunosorbent assay and is directed to GII.17 virus-likes The antibody of particle.In short, GII.17 virus-like particles are coated with 96 orifice plates, per hole 30ng, 4 DEG C of coatings overnight, are taken off with containing 5% The PBST of fat milk is closed, and is added 50ul hybridoma culture fluids to be incubated at 37 DEG C per hole 2 hours, is then incubated with the HRP secondary antibodies marked It educates 1 hour, finally carries out chromogenic reaction, read the light absorption value of OD450.

3. ascites prepares and antibody purification

Female Balb/c mouse peritoneals inject 500ul saxols, after two weeks, every mouse peritoneal inject 300,000 it is miscellaneous Hand over oncocyte.After 7 days, No. 12 syringe needles collect ascites, and 10,000rpm centrifugation 10min remove upper layer grease and lower sediment, take Clear ascites carries out antibody purification.According to specification, HiTrap HiTrapTM Protein G affinity columns (GE is utilized Health care) purifying ascites acquisition antibody.

4. enzyme-linked immunosorbent assay identifies monoclonal antibody

It is coated with 96 hole Elisa plates overnight with 4 DEG C of every hole 50ngGI.1 or GII.4 or GII.17 virus-like particles, identification is single Anti- binding ability.Elisa plates, will be different by every hole 50ul after the PBST containing 5% skim milk closes 1 hour at 37 DEG C Concentration (5ug/ml, 1ug/ml, 0.2ug/ml and 0.04ug/ml) is added 37 DEG C of monoclonal antibody and is incubated 2 hours, then with HRP labels Anti- mouse secondary antibody is incubated, and light absorption value OD450 is last read.

5. polyacrylamide gel electrophoresis and western blot analyses

After protein sample is mixed with SDS-PAG sample-loading buffers, processing 10min is boiled, through 12% polyacrylamide gel Protein isolate sample.Protein band is shown by coomassie brilliant blue staining or will be carried out on protein delivery to pvdf membrane Western blot analyses.Monoclonal antibody is diluted to by ultimate density 1ug/ml in the PBST containing 1% skim milk.Mouse is anti- GII.17 polyclonal antibodies 1:1000 dilutions use, and are then incubated with the HPR mouse secondary antibodies marked, are finally sent out with LAS-400 Light image analyzer is recorded.

6. sandwich Elisa detects GII.17 virus-like particles

With the polyvalent antibody 1 of rabbit-anti GII.17 virus-like particles:4 DEG C of 2000 dilutions (holes 50ul/) are coated with 96 holes overnight After the PBST containing 5% skim milk closes 2 hours at 37 DEG C Elisa is added in virus-like particle by Elisa plates, Elisa plates In plate, 40ng/50ul/ begins in hole, 2 concentration of doubling dilution 12,37 DEG C of 2 hours of incubation, then that virus-like particle is special 37 DEG C of holes monoclonal antibody 50ng/50ul/ be incubated 1 hour, be then incubated with the HPR mouse secondary antibodies marked, last read light absorption value OD450。

7. external substitute neutralizes experiment

96 hole Elisa plates are coated with PGMIII (holes 50ul/) room temperature of 10ug/ml, Elisa plates, which pass through, contains 5% skim milk PBST closed at 4 DEG C overnight after it is spare.GII.17 virus-like particle specific monoclonal antibodies 8ug/ml is begun, 2 doubling dilutions, with The GII.17 virus-like particles of isometric 0.5ug/ml are added to the 96 hole Elisa for being coated with PGMIII after being incubated at room temperature 1 hour On plate, it is incubated at room temperature 1 hour, rabbit-anti GII.17VLP polyvalent antibodies 1 are then added:1000 37 DEG C of dilutions are incubated 1 hour, Then it is incubated with the HPR rabbit secondary antibodies marked, last reads light absorption value OD450.

8. the gene order of monoclonal antibody expands and the structure of expression vector

The cell of hybridoma cell strain Trizol reagents are first extracted into total serum IgE, then according to 5 ' RACE kit explanations Book amplifies heavy chain and light chain full-length gene.It is introduced respectively at the 5 ' ends and 3 ' ends of heavy chain and light chain using the method for PCR amplification HindIII and EcoRI restriction enzyme sites, and the full gene of the heavy chain amplified and light chain is cloned into pGEM-T respectively (Promage) in, positive colony sequencing is filtered out, then sequence is correctly cloned and uses HindIII and EcoRI double digestions, warp After agarose gel electrophoresis is purified into target fragment, connect with plasmid pcDNA3.1 (Promage) the T4DNA ligases of same digestion It connects, is built into eukaryotic expression vector pcDNA3.1-(m3A3-3H) and pcDNA3.1- (m3A3-3L).

9. the recombinant expression of monoclonal antibody gene is identified

It is thin using the method cotransfection pcDNA3.1- (m3A3-3H) and pcDNA3.1- (m3A3-3L) Dao 293T of liposome Born of the same parents are collected culture supernatant after 72 hours and are analyzed, the expression of the antibody in culture supernatant is determined using ELISA：Use GII.17 Virus-like particle wrapper sheet is closed 1 hour in 37 DEG C with the PBST containing 5% milk, the culture supernatant to be measured of different dilutions is added 37 DEG C are incubated 2 hours, are then incubated with the HRP anti-mouse IgG secondary antibodies marked, last read light absorption value OD450.

Embodiment 1 secretes the screening of the hybridoma of GII.17 specific antibodies

The spleen cell that GII.17 virus-like particle mouse have been immunized is used for preparing hybridoma.It is tested by Elisa Hybridoma supernatant is screened, can secrete the hybridoma in conjunction with GII.17 virus-like particle abilities to obtain Strain.Finally, four plants of monoclonal antibodies are screened out, they can combine GII.17 virus-like particles.Subtype identification shows, 1D3-2, The heavy chain of 2C1-1,3A3-3 and 4B1-2 are IgG1, light chain kappa.

Table 1. secretes the hybridoma cell strain identification of monoclonal antibody

Sample for analysis is 50ul hybridoma culture cell conditioned mediums.

*,+：OD450 > 0.15；++：OD450 > 0.3；+++：OD450 > 0.5.

The specificity analysis of 2 anti-GII.17 monoclonal antibodies of embodiment

The purity and integrality of the GII.4 monoclonal antibodies purified from ascites by SDS-PAGE identifications first.Fig. 1 shows four The heavy chain and light chain of kind monoclonal antibody are respectively 50KD and 25KD or so.Then, monoclonal antibody and not synantigen are had detected by Elisa methods Reactivity, including GI.1 virus-like particles, GII.4 virus-like particles and GII.17 virus-like particles.Fig. 2 displays 1D3-2, 2C1-1,3A3-3 and 4B1-2 can with specific recognition GII.17 virus-like particles, and cannot identify GI.1 virus-like particles and GII.4 virus-like particles.Finally, the combination situation of monoclonal antibody and GI.1, GII.4 and GII.17 is analyzed by Western blot, Fig. 3 is shown, in four kinds of antibody：1D3-2,2C1-1 and 4B1-2 cannot identify GI.1, GII.4 and GII.17 virus after denaturation Sample particle, and 3A3-3 can identify GI.1, GII.4 and GII.17 virus-like particle after denaturation.

Sandwich Elisa of the embodiment 3 based on monoclonal antibody specifically can delicately detect GII.17 virus-like particles

It is measured by sandwich Elisa and monoclonal antibody (as OD450 > 0.15, sentences the minimum detectability degree of virus-like particle For the positive).Fig. 4 shows that 1D3-2,2C1-1,3A3-3 and 4B1-2 monoclonal antibody specifically can delicately detect GII.17 virus-likes Particle, minimum detectability degree are respectively 0.078ng, 0.078ng, 0.156ng, 0.156ng, prompt to can be used for GII.17 infection Diagnosis.

The potential neutralization activity of 4 monoclonal antibody of embodiment

Tissue blood group antigens (HBGA) are expression and the carbohydrate on mucosal tissue and red blood cell, are needed for norovirus infection Receptor.The combination of HBGA inhibits experiment to be widely used as the replacement neutralization test for antibody-mediated norovirus.Pig stomach is viscous Contain HBGA in liquid element III, has been verified and can be used for substituting neutralization test.By substitute neutralization test respectively to 1D3-2, The potential neutralization activity of tetra- kinds of monoclonal antibodies of 2C1-1,3A3-3 and 4B1-2 is detected.Fig. 5 shows that 1D3-2 and 3A3-3 are to GII.17 With stronger potential neutralization activity, can inhibit the EC50 that virus-like particle is combined with PGMIII is respectively：0.050ug/ml and 0.059ug/ml。

The gene sequencing of 5 monoclonal antibody of embodiment

Heavy chain and the sequence of light chain for cloning the monoclonal antibody of the 3A3-3 come are following (wherein,Single underscorePart is signal peptide sequence Row, italicized item is variable region sequences,Dotted line underscoreFor constant-region sequences)：

3A3-3 monoclonal antibody heavy chain nucleotide sequences：

3A3-3 monoclonal antibody heavy chain amino acid sequences：

3A3-3 monoclonal antibody light chain nucleotide sequences：

3A3-3 monoclonal antibody light-chain amino acid sequences：

Using online tool IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/), in Organism For query sequence are further analyzed under conditions of being Mouse, 3A3-3 monoclonal antibodies heavy chain variable region and light chain variable region Sequence, 3A3-3 monoclonal antibodies heavy chain variable amino acid are following (underscore mark is heavy chain CDR region)：

QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKLRPGHGLEWIGEILPGNDNSNYNKKFKGKATFTADTSSN TAYIQLGSLTSEDSAVYYCARSTWDKGYYYPLDYWGQGTSVTV(SEQ ID NO.4)

Above-mentioned heavy chain variable region belongs to IGHV1 subgroups.

3A3-3 monoclonal antibodies chain variable region amino acid is following (underscore mark is heavy chain CDR region)：

QIVLTQSPAIMSASPGEKVTLTCSASSSINYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSM EAEDAATYHCHQRSSSPWTFGGGTELEIK(SEQ ID NO.8)

Above-mentioned light chain variable region belongs to IGKV4 subgroups.

Each CDR region amino acid sequence and nucleotide sequence are summarized in table 2.

Table 2

The recombinant expression of 6 monoclonal antibody gene of embodiment and identification

Whether the gene in order to determine cloned 3A3-3 monoclonal antibodies is correct, and the coded sequence of heavy chain and light chain is distinguished It is inserted into pcDNA3.1, construction of expression vector pcDNA3.1- (m3A3-3H) and pcDNA3.1- (m3A3-3L), then corotation 293T cells are contaminated, and whether detected by ELISA has the antibody of specific binding GII.17 virus-like particles to deposit in cell conditioned medium ?.Fig. 5 shows, the cell conditioned medium of expression 3A3-3 monoclonal antibody sequences has very high binding signal, and OD450 values when being diluted at 8 times Just there is reduction slightly；The supernatant of control cell without transfecting related plasmids is all not bound with signal when not diluting.It should As a result it is the gene of 3A3-3 monoclonal antibodies to illustrate the sequence for expanding and expressing really.

It discusses

It is to obtain the monoclonal antibody of energy specific bond GII.17 virions that this, which studies initial purpose, further to use Such as sick kit of promise is diagnosed to develop, but surprisingly obtains two plants of monoclonal antibodies with powerful potential neutralization activity 1D3-2 and 3A3-3, this two plants of monoclonal antibodies can be used as therapeutic monoclonal antibodies drug after humanization in the future.It is logical Cross sandwich ELISA, the minimum detectability of monoclonal antibody 1D3-2,2C1-1,3A3-3 and 4B1-2 to GII.17 virus-like particles Degree is respectively 0.078ng, 0.078ng, 0.156ng, 0.156ng, this is to develop these monoclonal antibodies to examine at norovirus Test agent box provides advantageous theoretical foundation.It is obtained for the monoclonal antibody 1D3-2 of GII.17 and 3A3-3 pairs The EC50 of the interaction of GII.17 virus-like particles and PGMIII is respectively：0.050ug/ml and 0.059ug/ml.

In our current research, elaborate to be detected the feasibility of GII.17 with monoclonal antibody.Elisa experiments show these lists Clonal antibody can effectively detect GII.17 virus-like particles.

In conclusion available data is shown, the monoclonal antibody that the present invention screens acts not only as the useful detection work in laboratory Tool, and develop the reliable candidate of the effective material and the therapeutic monoclonal antibody of the anti-norovirus of humanization of diagnostic method.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

<110>Institut Pasteur of Shanghai, Chinese Academy of Sciences

<120>The preparation and application of anti-norovirus GII.17 monoclonal antibodies

<130> P2017-0090

<160> 18

<170> PatentIn version 3.5

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Ser Ser Ile Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Thr Ser

50 55 60

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro

65 70 75 80

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

85 90 95

Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr His Cys His Gln Arg

100 105 110

Ser Ser Ser Pro Trp Thr Phe Gly Gly Gly Thr Glu Leu Glu Ile Lys

115 120 125

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

130 135 140

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

145 150 155 160

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

165 170 175

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

180 185 190

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

195 200 205

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

210 215 220

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210> 13

<211> 24

<212> DNA

<213>Artificial sequence

<400> 13

ggctacacat tcagtagcta ctgg 24

<210> 14

<211> 24

<212> DNA

<213>Artificial sequence

<400> 14

attttacctg gaaatgataa ttct 24

<210> 15

<211> 45

<212> DNA

<213>Artificial sequence

<400> 15

gcaagatcta cctgggacaa gggttattac tatcctttgg actac 45

<210> 16

<211> 15

<212> DNA

<213>Artificial sequence

<400> 16

tcaagtataa attac 15

<210> 17

<211> 9

<212> DNA

<213>Artificial sequence

<400> 17

gacacatcc 9

<210> 18

<211> 27

<212> DNA

<213>Artificial sequence

<400> 18

catcagcgga gtagttcccc ctggacg 27

Claims (10)

1. a kind of heavy chain variable region of antibody, which is characterized in that the heavy chain variable region has below one or more mutual It mends and determines area CDR：

CDR1 shown in SEQ ID NO.1,

CDR2 shown in SEQ ID NO.2, and

CDR3 shown in SEQ ID NO.3.

2. a kind of heavy chain of antibody, which is characterized in that the heavy chain has heavy chain variable region described in claim 1 and heavy chain Constant region.

3. a kind of light chain variable region of antibody, which is characterized in that the light chain variable region has complementary determining region selected from the group below CDR：

CDR1 ' shown in SEQ ID NO.5,

CDR2 ' shown in SEQ ID NO.6, and

CDR3 ' shown in SEQ ID NO.7.

4. a kind of light chain of antibody, which is characterized in that the light chain has light chain variable region and light chain described in claim 3 Constant region.

5. a kind of antibody, which is characterized in that the antibody has：

(1) heavy chain variable region as described in claim 1；And/or

(2) light chain variable region as claimed in claim 3.

Preferably, the antibody has：Heavy chain as claimed in claim 2；And/or the light chain described in claim 4.

6. a kind of recombinant protein, which is characterized in that the recombinant protein has：

(i) sequence, the sequence of heavy chain as claimed in claim 2, such as right of heavy chain variable region as described in claim 1 are wanted Ask the sequence of the light chain variable region described in 3, the sequence of light chain as claimed in claim 4 or antibody as claimed in claim 5 Sequence；

(ii) polypeptide, protein drug sequence；And

(iii) sequence label of optional assistance expression and/or purifying.

7. a kind of polynucleotides, which is characterized in that it encodes polypeptide selected from the group below：

(1) sequence, the sequence of heavy chain as claimed in claim 2, such as right of heavy chain variable region as described in claim 1 are wanted Ask the sequence of the light chain variable region described in 3, the sequence of light chain as claimed in claim 4 or antibody as claimed in claim 5 Sequence；Or

(2) recombinant protein as claimed in claim 6.

8. a kind of carrier, which is characterized in that it contains the polynucleotides described in claim 7.

9. a kind of genetically engineered host cell, which is characterized in that it contains in carrier or genome according to any one of claims 8 It is integrated with the polynucleotides described in claim 7.

10. a kind of immune conjugate, which is characterized in that the immune conjugate contains：

(a) sequence, the sequence of heavy chain as claimed in claim 2, such as right of heavy chain variable region as described in claim 1 are wanted Ask the sequence of the light chain variable region described in 3, the sequence of light chain as claimed in claim 4, antibody as claimed in claim 5 Sequence or recombinant protein as claimed in claim 6；With

(b) coupling moiety selected from the group below：Detectable marker, drug, toxin, cell factor, radionuclide or enzyme.