CN105153304B - The broad-spectrum monoclonal antibody of anti-HPV L1 albumen or its antigen-binding fragment and their purposes - Google Patents

Abstract

The present invention relates to can wide spectrum combination human papilloma virus (HPV) L1 albumen monoclonal antibody and its antigen-binding fragment, and encode their sequence, generate their cell strain, and the method and purposes for being diagnosed, being prevented or being treated using them.

Description

The broad-spectrum monoclonal antibody of anti-HPV L1 albumen or its antigen-binding fragment and they Purposes

The application be the applying date be on June 8th, 2012, the entitled " broad-spectrum monoclonal antibody of anti-HPV L1 albumen Or its antigen-binding fragment and their purposes ", application No. is the divisional applications of 201210190399.5 application.

Technical field

The present invention relates to Molecular Virology and field of immunology.Specifically, the present invention relates to can wide spectrum combination human milk head The monoclonal antibody and its antigen-binding fragment of tumor virus (HPV) L1 albumen, and their sequence is encoded, generate the thin of them Born of the same parents' strain, and the method and purposes that are diagnosed, prevented or treated using them.

Background technology

Human papilloma virus (HPV) is a kind of nonenveloped virus, it is by viral capsid and the about 8kb being wrapped in capsid DNA is constituted.The viral capsid of HPV is by Major capsid protein L1 and secondary capsid protein the L2 a diameter of 50-55nm's formed Regular dodecahedron particle.The HPV of more than 100 kinds of type is had now been found that, wherein can be with tumorigenic relationship according to it It is divided into two classes：Low risk HPV-- includes HPV6 and HPV11, and carcinogenic risk is low, mainly causes condyloma acuminatum；High-risk-type HPV-- includes HPV16,18,31,33,35,39,45,52,58,59 etc., be cause it is more including women cervical carcinoma The main reason for kind of tumor disease (Clifford GM, Smith JS, Plummer M, et a1.Br J Cancer, 2003,88 (1)：63-73).Existing result of study shows that the generation of the diseases such as cervical carcinoma can be prevented by preventing HPV infection.

The vaccine research of HPV finds that the HPV L1 albumen of vivoexpression can be self-assembled into virus-like particle (VLP), ties Structure is similar to natural HPV height, remains most neutralizing epitopes of natural viral, can induce the neutralizing antibody of high titre. Therefore, the existing and HPV vaccines researched and developed are mostly with virus-like particle for main vaccine composition.However, the study found that HPV VLP mainly induce the neutralizing antibody for homotype HPV, generate the protective immunity for homotype HPV, and only same at some There are cross protection (Giroglou T, Sapp M, Fligge C, et al.Vaccine.2001 between the high type of source property；19 (13-14):1783-93；Orozco JJ,Carter JJ,Koutsky LA.J Virol[J].2005；79(15):9503- 14；Fleury MJ, Touze A, Maurel MC, et al.Protein Sci.2009.18 (7):1425-1438).

In vaccine research, monoclonal antibody is the important tool of vaccine antigen Quality Control, and antibody level is evaluation vaccine effect The standard of fruit, moreover, antibody and its epitope (the especially corresponding neutralizing epitope of neutralizing antibody) and corresponding neutralization mechanism It is the important pointer of vaccine research and development.Further, in application study, neutralizing antibody, especially wide spectrum neutralizing antibody, HPV's It diagnoses, will also have special advantage in prevention and treatment.However, existing studies have shown that the antibody that HPV L1 albumen is induced It is mostly type specificity neutralizing antibody, rarely across the report of the wide spectrum neutralizing antibody of type.At present, it has been found that across type Antibody only have HPV16J4, HPV16I23 and HPV33E12 this 3 plants of cross-neutralization monoclonal antibodies, they can have the HPV of several types There is a degree of cross-neutralization.But the dilution factor of this three plants of monoclonal antibodies is relatively low, (such as than specific neutralization monoclonal antibody： Neutralizing antibody V5, referring to Wang etc., Journal of General irology, 78：2209-2215 (1997)) it is 100 times small More than, which greatly limits the applications of these monoclonal antibodies.

Therefore, this field still needs more, more effective wide spectrum antibody, especially wide spectrum neutralizing antibody, with can be right The vaccine of production and the protecting effect of vaccine are preferably evaluated, and are applied in the diagnosis of HPV, prevention and treatment.

Invention content

The present invention provides can wide spectrum combination HPV L1 albumen monoclonal antibody and its antigen-binding fragment, and coding Their sequence generates their cell strain, and the method and purposes for being diagnosed, being prevented or being treated using them.

In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology The normally understood meaning of personnel institute.Also, cell culture used herein, molecular genetics, nucleic acid chemistry, immunological experiment Room operating procedure is widely used conventional steps in corresponding field.Meanwhile for a better understanding of the present invention, it is provided below The definition and explanation of relational language.

As used herein, term " HPV L1 albumen " refers to that the L1 albumen of human papilloma virus (HPV) is It is well known in the art (see, e.g. NCBI GENBANK database serial numbers：DQ469930.1).In this application, when referring to When the L1 albumen or VLP of HPV, the L1 albumen or VLP of the HPV selected from following 11 kinds of types are related generally to：HPV6、HPV11、 HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52, HPV58 and HPV59.However, those skilled in the art manage Solution, term " HPV L1 albumen " can also include the L1 albumen of the HPV of other types.

In this application, for convenience's sake, when describing the amino acid sequence of HPV L1 albumen, unless context is special It points out, otherwise, is described with reference to the amino acid sequence of HPV16L1 albumen.For example, statement " the 303-308 of HPV L1 albumen Amino acids residue " refers to the 303-308 amino acids residues of HPV16L1 albumen.However, as used herein, art Language " HPV L1 albumen " be intended to include the other HPV of all models (especially above-mentioned 11 kinds of types) L1 albumen.Therefore, " HPV is stated The 303-308 amino acids residue of L1 albumen " not only includes the 303-308 amino acids residues of HPV16L1 albumen, but also Corresponding sequence segment in HPV L1 albumen including other types.

As used herein, when referring to the amino acid sequence of HPV16L1 albumen, with reference to SEQ ID NO:59 ammonia Base acid sequence is described.For example, statement " the 303-308 amino acids residue of HPV16L1 albumen " refers to SEQ ID NO:The 303-308 amino acids residues of amino acid sequence shown in 59.However, it will be appreciated by those skilled in the art that in HPV16L1 In the amino acid sequence of albumen, can it is naturally-produced or be artificially introduced mutation or variation (include but not limited to, displacement, missing and/or Addition), without influencing its biological function.Therefore, in the present invention, term " HPV16L1 albumen " should include all such sequences Row, including such as SEQ ID NO:Sequence shown in 59 and its natural or artificial variant.Also, when description HPV16L1 eggs Include not only SEQ ID NO when white sequence fragment:59 sequence fragment further includes SEQ ID NO:59 natural or people Corresponding sequence segment in work variant.For example, statement " the 303-308 amino acids residue of HPV 16L1 albumen " includes SEQ ID NO:Respective segments in 59 303-308 amino acids residue and its variant (natural or artificial).According to this hair Bright, statement " corresponding sequence segment " or " respective segments " refers to, when carrying out optimal comparison to sequence, i.e., when sequence is compared When obtaining highest percentage homogeneity, the segment of equivalent site is located in the sequence being compared.

As used herein, term " antibody " refer to refer to usually (each pair of to have one " light " by two pairs of polypeptide chains (L) chain and " weight " (H) chain) composition immunoglobulin molecules.Antibody light chain can be classified as κ and lambda light chain.Heavy chain can classify For μ, δ, γ, α or ε, and the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE respectively.In light chain and heavy chain Interior, variable region is connected with constant region by area " J " of about 12 or more amino acid, and heavy chain also includes about 3 or more Area " D " of a amino acid.Each heavy chain is by heavy chain variable region (V_H) and heavy chain constant region (C_H) composition.Heavy chain constant region is by 3 structures Domain (C_H1、C_H2 and C_H3) it forms.Each light chain is by light chain variable region (V_L) and constant region of light chain (C_L) composition.Constant region of light chain is by one A domain C_LComposition.The constant region of antibody can mediated immunity globulin and host tissue or the factor, including immune system is each The combination of the first component (C1q) of kind cell (for example, effector cell) and classical complement system.V_HAnd V_LArea can be also subdivided into With denatured region (being known as complementary determining region (CDR)), it is interspersed with the more conservative region for being known as framework region (FR). Each V_HAnd V_LBy in the following order：FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 arranged from amino terminal to carboxyl terminal 3 A CDR and 4 FR compositions.Variable region (the V of each heavy chain/light chain pair_HAnd V_L) it is respectively formed paratope.Amino acid is to each The distribution of region or structural domain follows Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)) or Chothia＆Lesk (1987)J.Mol.Biol.196:901-917；Chothia et al. (1989) Nature 342:The definition of 878-883.Term " antibody " is not limited by any specific method for generating antibody.Such as comprising, particularly, recombinant antibodies, monoclonal antibody And polyclonal antibody.Antibody can be the antibody of different isotypes, for example, IgG is (for example, IgG1, IgG2, IgG3 or IgG4 are sub- Type), IgA1, IgA2, IgD, IgE or IgM antibody.

As used herein, " antigen-binding fragment " of term antibody refers to the more of the segment comprising full length antibody Peptide keeps the ability for specifically binding the same antigen that full length antibody is combined, and/or is competed to antigen with full length antibody Specific binding, also referred to as " antigen-binding portion thereof ".Usually referring to Fundamental Immunology, Ch.7 (Paul, W., ed., second edition, Raven Press, N.Y. (1989) are incorporation by reference in its entirety, are used for institute Purposefully.The antigen-binding fragment of antibody can be generated by recombinant DNA technology or by the enzymatic or chemical disruption of complete antibody. In some cases, antigen-binding fragment includes Fab, Fab', F (ab')₂, Fd, Fv, dAb and complementary determining region (CDR) segment, Single-chain antibody (for example, scFv), chimeric antibody, double antibody (diabody) and such polypeptide, it includes be enough to assign polypeptide spy At least part of the antibody of Specific Antigen binding ability.

As used herein, term " Fd segments " means by V_HAnd C_HThe antibody fragment of 1 structural domain composition；Term " Fv Segment " means by the V of the single armed of antibody_LAnd V_HThe antibody fragment of structural domain composition；Term " dAb segments " means by V_HStructural domain Antibody fragment (Ward et al., the Nature 341 of composition:544-546(1989))；Term " Fab segments " means by V_L、V_H、C_L And C_HThe antibody fragment of 1 structural domain composition；Term " F (ab')₂Segment " means comprising by the disulphide bridges connection on hinge area The antibody fragment of two Fab segments.

In some cases, the antigen-binding fragment of antibody is single-chain antibody (for example, scFv), wherein V_LAnd V_HStructural domain Connector by the way that single polypeptide chain can be produced as match to be formed monovalent molecule (see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc.Natl.Acad.Sci.USA 85:5879-5883 (1988)).Such scFv molecules can have general structure：NH₂-V_LConnector-V_H- COOH or NH₂-V_HConnector-V_L-COOH.It closes Suitable prior art connector is made of the GGGGS amino acid sequences repeated or its variant.For example, can be used has amino acid sequence (GGGGS)₄Connector, but its variant (Holliger et al. (1993), Proc.Natl.Acad.Sci.USA 90 can also be used: 6444-6448).Other connectors for use in the present invention are by Alfthan et al. (1995), Protein Eng.8:725-731, Choi et al. (2001), Eur.J.Immunol.31:94-106, Hu et al. (1996), Cancer Res.56:3055-3061, Kipriyanov et al. (1999), J.Mol.Biol.293:41-56 and Roovers et al. (2001), Cancer Immunol. Description.

In some cases, the antigen-binding fragment of antibody is double antibody, that is, bivalent antibody, wherein V_HAnd V_LStructural domain exists It is expressed in single polypeptide chain, but using too short connector so that do not allow to match between two structural domains of same chain, from And force the complementary domain of structural domain and another chain to match and generate two antigen-binding sites (see, e.g., Holliger P. et al., Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993) and Poljak R.J. et al., Structure 2:1121-1123(1994))。

It can be used routine techniques well known by persons skilled in the art (for example, recombinant DNA technology or enzymatic or chemical disruption Method) antigen of antibody is obtained from given antibody (such as monoclonal antibody 4B3,13A10,12B9 or 4H4 provided by the invention) Binding fragment (for example, above-mentioned antibody fragment), and in mode identical with the mode for complete antibody with regard to specificity screening The antigen-binding fragment of antibody.

Herein, unless clearly indicated by the context, include not only complete anti-otherwise when referring to term " antibody " Body, and include the antigen-binding fragment of antibody.

As used herein, term " monoclonal antibody " and " monoclonal antibody " refer to the antibody from a group very high homology One segment of an antibody or antibody in molecule, namely in addition to the natural mutation of possible spontaneous appearance, a group is identical Antibody molecule.Monoclonal antibody has high specific to the single epitope on antigen.Polyclonal antibody be relative to monoclonal antibody and Speech, at least two kinds of or more different antibodies are generally comprised, the different tables on these the generally recognized antigens of different antibody Position.Monoclonal antibody usually can be used the hybridoma technology that Kohler etc. reports for the first time obtain (Nature, 256:495,1975), But recombinant DNA technology can also be used and obtain (such as referring to U.S.P 4,816,567).

As used herein, the Dan Ke to number the monoclonal antibody referred to be obtained from the hybridoma of identical number Grand antibody is identical.For example, monoclonal antibody 4B3 (or 13A10,12B9 or 4H4) be respectively with from hybridoma cell strain 4B3 (or 13A10,12B9 or 4H4) or it is subcloned or the identical antibody of antibody of progeny cell acquisition.

As used herein, term " chimeric antibody " refers to a part for such antibody, light chain or/and heavy chain From an antibody (it can be originated from a certain particular species or belong to a certain specific antibodies class or subclass), and light chain or/and again Another part of chain is originated from another antibody, and (it can be originated from identical or different species or belong to identical or different antibody class Or subclass), nevertheless, it still retains the activity of the combination to target antigen (U.S.P 4,816,567to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:68516855(1984)).

As used herein, term " humanized antibody " refers to the whole of people source immunoglobulin (receptor antibody) Or the antibody or antibody fragment that part CDR region is obtained after the CDR region replacement of one non-human source antibodies (donor antibody), confession therein Body antibody can have expected specificity, compatibility or reactive inhuman source (for example, mouse, rat or rabbit) antibody.This Outside, some amino acid residues of the framework region (FR) of receptor antibody can also be replaced by the amino acid residue of corresponding non-human source antibodies It changes, or is replaced by the amino acid residue of other antibody, further to improve or optimize the performance of antibody.About humanized antibody More detailed contents, reference can be made to for example, Jones et al., Nature, 321:522 525(1986)；Reichmann et al.,Nature,332:323 329(1988)；Presta,Curr.Op.Struct.Biol.,2:593 596(1992)；With Clark,Immunol.Today 21:397 402(2000)。

As used herein, " neutralizing antibody " refer to can remove or significantly reduce target viral virulence (for example, The ability of infection cell) antibody or antibody fragment.

As used herein, term " epitope " refers to being combined by immunoglobulin or antibody specificity on antigen Position." epitope " is also referred to as " antigenic determinant " in the art.Epitope or antigenic determinant are usually by the chemism of molecule Surface group such as amino acid or carbohydrate or carbohydrate side chain form and usually have specific three-dimensional structural feature and Specific charge characteristic.For example, epitope includes usually at least 3,4,5,6,7,8,9,10,11,12 with unique space conformation, 13,14 or 15 amino acid continuously or discontinuously can be " linear " or " conformation ".See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, volume 66, G.E.Morris, Ed. (1996).In linear epitope, the points of all interactions between protein and interacting molecule (such as antibody) along The primary amino acid sequences of protein linearly exist.In comformational epitope, the point of interaction crosses over the protein being separated from each other Amino acid residue and exist.

As used herein, term " epitope peptide " refers to that can be used as the peptide fragment of epitope on antigen.In some cases Under, individual epitope peptide can be by antibody specificity identification/combination for the epitope.In other cases, may It needs to merge epitope peptide with carrier protein, so that epitope peptide can be identified by specific antibody.As used herein, art Language " carrier protein " refers to such albumen, can serve as the carrier of epitope peptide, that is, it can be inserted into table in specific location Position peptide, so that the epitope peptide can show, to which the epitope peptide can be identified by antibody or immune system.Examples of such carriers egg It is well known to those skilled in the art in vain, including for example, epitope peptide (can be inserted in the of the albumen by HPV L1 albumen Between 127-128 amino acids, or between 423-424 amino acids, see, for example, Varsani A, Chimeric human papillomavirus type 16(HPV-16)L1particles presenting the common neutralizing epitope for the L2minor capsid protein of HPV-6and HPV-16.J Virol.2003Aug；77(15):8386-93), HbcAg (can replace 79-81 of the albumen with epitope peptide Amino acid, referring to Schodel, F.The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity.J Virol, 1992,66:106-114) and epitope peptide (can be connected to the N-terminal or the ends C of the albumen or its segment by CRM197 albumen End).

In the present invention, CRM197 (Cross-Reacting Materials 197) refers to the one of diphtheria toxin (DT) Kind non-toxic mutant (Uchida, T., A.M, Pappenheimer, Jr., R.Gregory, et al., J.Biol.Chem.1973.248:3838-3844), compared with the wild type gene of encoding D T there are single nucleotide acid mutation, Cause the 52nd amino acid residue from Gly become Glu (G.Giannini, R.Rappuoli, G.Rattiet al., Nucleic Acids Research.1984.12：4063-4070).

Routine techniques well known by persons skilled in the art can be used, just with the binding competition screening antibodies of same epitope. For example, can be at war with and cross competition research, to be contended with one other or cross competition and antigen (for example, HPV L1 albumen) Combination antibody.Based on their cross competition the world is described in obtain the high throughput method of the antibody in conjunction with same epitope In patent application WO 03/48731.Therefore, routine techniques well known by persons skilled in the art can be used, obtain with the present invention's Same epitope on monoclonal antibody (for example, monoclonal antibody 4B3,13A10,12B9 or 4H4) competitive binding L1 albumen it is anti- Body and its antigen-binding fragment (that is, antigen-binding portion thereof).

As used herein, term " separation " or " separation " refer under native state through artificial hand What section obtained.If occurring the substance or ingredient of a certain " separation " in nature, it would be possible that being the natural surroundings residing for it Changed, or isolate the substance or the two situation under natural surroundings to have generation.For example, a certain living animal body The interior naturally occurring polynucleotide or polypeptide that certain is not detached, and the high-purity separated under this native state Identical polynucleotide or polypeptide are to be referred to as separation.Term " separation " or " separation " be not excluded for being mixed with it is artificial or The substance of synthesis does not exclude the presence of not the other foreign bodys for influencing species activity yet.

As used herein, term " escherichia expression system " refers to being made of with carrier Escherichia coli (bacterial strain) Expression system, wherein Escherichia coli (bacterial strain) derive from bacterial strain available on the market, such as, but not limited to：GI698, ER2566,BL21(DE3),B834(DE3),BLR(DE3)。

As used herein, term " carrier (vector) " refers to the one kind that can be inserted polynucleotide Nucleic acid delivery vehicle.When carrier can make the albumen of the polynucleotide encoding of insertion obtain expression, carrier is known as expression vector.It carries Body can import host cell by conversion, transduction or transfection, and the inhereditary material element that it is carried is made to be obtained in host cell It must express.Carrier is well known to those skilled in the art, including but not limited to：Plasmid；Phasmid；Coemid；It is artificially colored Body, for example, yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or the sources P1 artificial chromosome (PAC)；Phagocytosis Body such as bacteriophage lambda or M13 bacteriophages and animal virus etc..The animal virus that can be used as carrier includes but not limited to reverse transcriptase Viral (including slow virus), adenovirus, adeno-associated virus, herpesviral (such as herpes simplex virus), poxvirus, baculoviral, Papillomavirus, papova viruses (such as SV40).A kind of element that carrier can be expressed containing various control, including but It is not limited to, promoter sequence, transcriptional initiation sequence, enhancer sequence, selection element and reporter gene.In addition, carrier can also contain There is replication origin.

As used herein, term " host cell " refers to the cell that can be used for importing carrier comprising but it is unlimited In the fungal cell of, such as the prokaryotic cell of Escherichia coli or withered grass bacterium, such as yeast cells or Aspergillus, such as S2 drosophila cells Or the insect cell of Sf9 etc., or such as fibroblast, Chinese hamster ovary celI, COS cells, NSO cells, HeLa cells, bhk cell, The zooblast of 293 cells of HEK or people's cell etc..

As used herein, term " homogeneity " be used to refer between two polypeptides or between two nucleic acid sequence With situation.When some position in the sequence that two are compared all is occupied by identical base or amino acid monomer subunit (for example, some position in each of two DNA moleculars by adenine occupy or two polypeptides each in certain A position is all occupied by lysine), then each molecule is same on the position.Between two sequences " percentage is same Property " is the function of the matching position number shared by the two sequences divided by position number × 100 being compared.For example, such as There are 6 matchings in 10 positions of two sequences of fruit, then the two sequences have 60% homogeneity.For example, DNA sequence dna CTGACT and CAGGTT shares 50% homogeneity (having 3 location matches in 6 positions in total).In general, by two sequences It is compared when comparing to generate maximum homogeneity.Such comparison can be by using for example, can be by computer program for example Needleman that Align programs (DNAstar, Inc.) easily carry out et al. (1970) J.Mol.Biol.48：443-453's Method is realized.E.Meyers and the W.Miller (Comput.Appl for being integrated into ALIGN programs (version 2 .0) also can be used Biosci., 4:11-17 (1988)) algorithm, use PAM120 weight residues table (weight residue table), 12 Gap Length Penalty and 4 Gap Penalty measure the percentage homogeneity between two amino acid sequences.In addition, can be used The Needleman and Wunsch (J MoI being integrated into the GAP programs of GCG software packages (can be obtained on www.gcg.com) Biol.48:444-453 (1970)) algorithm, use 62 matrixes of Blossum or PAM250 matrixes and 16,14,12,10,8,6 Or 4 Gap Weight (gap weight) and 1,2,3,4,5 or 6 Length Weight measure hundred between two amino acid sequences Score homogeneity.

As used in this article, term " conservative substitution " means to influence or change comprising amino acid sequence The amino acid replacement of the necessary characteristic of protein/polypeptide.For example, standard technique known in the art such as direct mutagenesis can be passed through Conservative substitution is introduced with the mutagenesis that PCR is mediated.Conservative amino acid replacement includes being substituted with the amino acid residue with similar side chain The displacement of amino acid residue is used for example in physically or functionally similar with corresponding amino acid residue (such as with similar Size, shape, charge, chemical property include the ability etc. for forming covalent bond or hydrogen bond) the displacement that carries out of residue.At this The family of the amino acid residue with similar side chain is defined in field.These families include having basic side chain (for example, relying ammonia Acid, arginine and histidine), acid side-chain (such as aspartic acid, glutamic acid), uncharged polar side chain (such as sweet ammonia Acid, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar sidechain (such as third Propylhomoserin, valine, leucine, isoleucine, proline, phenylalanine, methionine), β branched building blocks (for example, threonine, Valine, isoleucine) and beta-branched side (for example, tyrosine, phenylalanine, tryptophan, histidine) amino acid.Cause This, preferably substitutes corresponding amino acid residue with another amino acid residue from same side chain family.Identify that amino acid is protected The method for keeping displacement is well known in the art (see, e.g., Brummell et al., Biochem.32:1180-1187 (1993)；Kobayashi et al. Protein Eng.12 (10):879-884(1999)；With Burks et al. Proc.Natl Acad.Set USA 94:412-417 (1997), is incorporated herein by reference).

As used in this article, term " immunogenicity (immunogenicity) " refers to that body can be stimulated to form spy The ability of xenoantibody or sensitized lymphocyte.It both referred to, and antigen can stimulate specific immunocyte, makes activated immune cell, increases It grows, break up, the final characteristic for generating immunologic effector substance such as antibody and sensitized lymphocyte, after also referring to antigenic stimulus body, machine Body immune system can form the specific immune response of antibody or sensitized T lymphocyte.Immunogenicity is the most important property of antigen Matter induces to a kind of antigen success host to generate the factor that immune response depends on three aspects：The property of antigen, host Reactivity and immunization ways.

As used in this article, term " specific binding " refers to two intermolecular nonrandom association reactions, such as antibody Reaction between its targeted antigen.In some embodiments, the antibody of certain antigen is specifically bound (or to certain antigen Antibody with specificity) refer to that antibody is to be less than about 10^-5M is, for example, less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10M or smaller affinity (K_D) combine the antigen.

As used herein, term " K_D" refer to specific antibodies-antigen interactions Dissociation equilibrium constant, use Binding affinity between description antibody and antigen.Equilibrium dissociation constant is smaller, and antibody-antigen binding is closer, antibody with Affinity between antigen is higher.In general, antibody (for example, monoclonal antibody 4B3,13A10,12B9 or 4H4 of the present invention) with Less than about 10^-5M is, for example, less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10M or smaller Dissociation equilibrium constants (K_D) In conjunction with antigen (for example, L1 albumen), for example, as measured in BIACORE instrument using surface plasma body resonant vibration art (SPR).

As used herein, " broad-spectrum monoclonal antibody of anti-HPV L1 albumen " and " wide spectrum combination HPV L1 are stated The monoclonal antibody of albumen " refers to, monoclonal antibody can a variety of types of specific recognition/combination (for example, at least four kinds of, such as At least five kinds of, at least six kinds of, at least seven kinds of, at least eight kinds of, at least nine kinds of, at least ten kinds of or 11 kinds of types) HPV L1 albumen.By It can be self-assembled into virus-like particle (VLP) in the HPV L1 albumen of vivoexpression, therefore, state " the wide spectrum of anti-HPV L1 albumen Monoclonal antibody " and " monoclonal antibody of wide spectrum combination HPV L1 albumen " also refer to, and monoclonal antibody being capable of specific recognition/knot Close a variety of types (it is for example, at least 5 kinds, at least six kinds of for example, at least four kinds of, it is at least seven kinds of, it is at least eight kinds of, it is at least nine kinds of, it is at least ten kinds of, Or 11 kinds of types) HPV VLP.For example, monoclonal antibody 4B3,13A10,12B9 or 4H4 of the present invention being capable of specificity knowledges The L1 albumen and VLP of the HPV of not/at least five kinds of types of combination.

As used herein, it term " monoclonal antibody " and " monoclonal antibody " meaning having the same and is used interchangeably； It term " polyclonal antibody " and " more anti-" meaning having the same and is used interchangeably；Term " polypeptide " and " protein " have phase With meaning and be used interchangeably.And in the present invention, amino acid is usually contracted with single-letter well known in the art and trigram It writes to indicate.For example, alanine can be indicated with A or Ala.

As used herein, term " hybridoma " and " hybridoma cell strain " are used interchangeably, and are worked as and referred to art Further include the subclone and progeny cell of hybridoma when language " hybridoma " and " hybridoma cell strain ".For example, when referring to hybridization When tumor cell strain 4B3, also refer to the subclone and progeny cell of hybridoma cell strain 4B3.

As used herein, term " pharmaceutically acceptable carrier and/or excipient " refer in pharmacology and/or The physiologically carrier and/or excipient compatible with subject and active constituent, be it is well known in the art (see, for example, Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company, 1995), and include but not limited to：PH adjusting agent, surface Activating agent, adjuvant, ionic strength reinforcing agent.For example, pH adjusting agent includes but not limited to phosphate buffer；Surfactant packet Include but be not limited to cation, anion or nonionic surface active agent, such as Tween-80；Ionic strength reinforcing agent includes But it is not limited to sodium chloride.

As used herein, term " adjuvant " refers to nonspecific immunity strengthening agent, when its together with antigen or in advance When first delivering into body, the immune response of body fight original can be enhanced or change type of immune response.There are many kinds of adjuvants, packet Include but be not limited to aluminium adjuvant (such as aluminium hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), short Corynebacterium, lipopolysaccharides, cell factor etc..Freund's adjuvant is most common adjuvant in current animal experiment.Aluminium hydroxide is helped Agent is then using more in clinical trial.

As used herein, term " HPV VLP " refers to being self-assembly of by the HPV L1 albumen of vivoexpression Virus-like particle.

As used herein, term " HPV pseudovirus " refers to that non-specific can pack nucleic acid using HPV VLP Characteristic by expressing L1 the and L2 albumen of HPV in the cell, and wraps up intracellular episome viral DNA or external source imports Reporter plasmid, and HPV pseudovirus (Yeager, M.D, Aste-Amezaga, M.et al (2000) Virology (278) formed 570-7).The method for being used to form pseudovirus includes for example, expression of recombinant virus systems approach and more plasmid co-transfection methods.This Signified HPV pseudovirus mainly includes the HPV pseudovirus of 11 types in invention, be respectively HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV33, HPV 35, HPV 45, HPV 52, HPV 58,59 pseudovirus of HPV.

As used herein, term " effective quantity " refers to being enough to obtain or at least partly obtain desired effect Amount.For example, preventing disease (such as HPV infection or with the relevant disease of HPV infection) effective quantity and referring to, it is sufficient to prevent, prevents, or Postpone the amount of the generation of disease (such as HPV infection or with the relevant disease of HPV infection)；Treating condition effective amount refers to, it is sufficient to Cure or at least partly prevent to have suffered from disease patient disease and its complication amount.Such effective quantity is measured completely to exist Within the limit of power of those skilled in the art.It will be depending on disease to be treated for example, effectively being measured for therapeutical uses Severity, patient oneself immune system overall status, the ordinary circumstance of patient such as age, weight and gender, drug Method of application, and the other treatment etc. that is administered simultaneously.

In one aspect, the present invention provides the HPV's that can specifically bind multiple types (for example, at least 4 types) The monoclonal antibody or its antigen-binding fragment of L1 albumen and/or VLP, wherein

The monoclonal antibody includes being selected from following heavy chain variable region (VH)：

(1) it is SEQ ID NO comprising amino acid sequence:The VH of the CDR of 17-19；

(2) it is SEQ ID NO comprising amino acid sequence:The VH of the CDR of 23-25；

(3) it is SEQ ID NO comprising amino acid sequence:The VH of the CDR of 29-31；With

(4) it is SEQ ID NO comprising amino acid sequence:The VH of the CDR of 35-37, and/or

The monoclonal antibody includes being selected from following light chain variable region (VL)：

(1) it is SEQ ID NO comprising amino acid sequence:The VL of the CDR of 20-22；

(2) it is SEQ ID NO comprising amino acid sequence:The VL of the CDR of 26-28；

(3) it is SEQ ID NO comprising amino acid sequence:The VL of the CDR of 32-34；With

(4) it is SEQ ID NO comprising amino acid sequence:The VL of the CDR of 38-40.

In a preferred embodiment, the monoclonal antibody includes being selected from following heavy chain variable region (VH)：

(1) such as SEQ ID NO:VH shown in 2；

(2) such as SEQ ID NO:VH shown in 6；

(3) such as SEQ ID NO:VH shown in 10；With

(4) such as SEQ ID NO:VH shown in 14.

In a preferred embodiment, the monoclonal antibody includes being selected from following light chain variable region (VL)：

(1) such as SEQ ID NO:VL shown in 4；

(2) such as SEQ ID NO:VL shown in 8；

(3) such as SEQ ID NO:VL shown in 12；With

(4) such as SEQ ID NO:VL shown in 16.

In a preferred embodiment, the monoclonal antibody includes：

(1) it is SEQ ID NO comprising amino acid sequence:The VH of the CDR of 17-19, and comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 20-22；

(2) it is SEQ ID NO comprising amino acid sequence:The VH of the CDR of 23-25, and comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 26-28；

(3) it is SEQ ID NO comprising amino acid sequence:The VH of the CDR of 29-31, and comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 32-34；Or

(4) it is SEQ ID NO comprising amino acid sequence:The VH of the CDR of 35-37, and comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 38-40.

In a preferred embodiment, the monoclonal antibody includes：

(1) such as SEQ ID NO:VH shown in 2, and such as SEQ ID NO:VL shown in 4；

(2) such as SEQ ID NO:VH shown in 6, and such as SEQ ID NO:VL shown in 8；

(3) such as SEQ ID NO:VH shown in 10, and such as SEQ ID NO:VL shown in 12；Or

(4) such as SEQ ID NO:VH shown in 14, and such as SEQ ID NO:VL shown in 16.

In a preferred embodiment, the monoclonal antibody be by hybridoma cell strain 4B3,4H4,12B9 or The monoclonal antibody that 13A10 is generated, described hybridoma cell strain 4B3,4H4,12B9 and 13A10 are preserved in Chinese Typical Representative culture Object collection (CCTCC), and it is respectively provided with preserving number CCTCC-C201264, CCTCC-C201265, CCTCC- C201266 and CCTCC-C201267.

In a preferred embodiment, the monoclonal antibody or its antigen-binding fragment are selected from Fab, Fab', F (ab')₂, Fd, Fv, dAb, complementary determining region segment, single-chain antibody (for example, scFv), humanized antibody, chimeric antibody or dual anti- Body.

In a preferred embodiment, the monoclonal antibody is to be less than about 10^-5M is, for example, less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10M or smaller K_DIn conjunction with the L1 albumen or VLP of HPV.

In a preferred embodiment, the monoclonal antibody can be specifically bound selected from following at least 5 Kind of type (it is at least seven kinds of for example, at least six kinds of, it is at least eight kinds of, it is at least nine kinds of, it is at least ten kinds of or 11 kinds) HPV L1 albumen and VLP：HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52, HPV58 and HPV59.At one In preferred embodiment, the monoclonal antibody can specifically bind at least HPV16, HPV31, HPV33, HPV35 and The L1 albumen and VLP of HPV52.

For example, the present invention monoclonal antibody 4B3 can specifically bind 11 kinds of types HPV L1 albumen and VLP；The present invention monoclonal antibody 13A10 can specifically bind 6 kinds of types HPV (HPV16, HPV31, HPV33, HPV35, HPV52 and HPV58) L1 albumen and VLP；The monoclonal antibody 4H4 of the present invention can specifically bind 5 kinds of types The L1 albumen and VLP of HPV (HPV16, HPV31, HPV33, HPV35 and HPV52)；The monoclonal antibody 12B9 of the present invention can be special The opposite sex combines the L1 albumen and VLP of the HPV (HPV16, HPV31, HPV33, HPV35, HPV52 and HPV58) of 6 kinds of types.

In a preferred embodiment, the monoclonal antibody includes non-CDR region, and the non-CDR region comes From the species for not being muroid, such as from human antibody.

In a preferred embodiment, the monoclonal antibody is neutralizing antibody, can neutralize at least two type Other HPV, such as at least two type in HPV16,31,33 and 58, at least three type or 4 types.

In a preferred embodiment, the monoclonal antibody can neutralize HPV16,31,33 and 58 4 types Other HPV comprising：Including amino acid sequence is SEQ ID NO:The VH of the CDR of 23-25, and/or be comprising amino acid sequence SEQ ID NO:The VL of the CDR of 26-28.Preferably, such monoclonal antibody includes, such as SEQ ID NO:VH shown in 6 and/or Such as SEQ ID NO:VL shown in 8.It is highly preferred that such monoclonal antibody is monoclonal antibody 13A10.

In a preferred embodiment, the monoclonal antibody can neutralize HPV33 and 58 two type HPV comprising：Including amino acid sequence is SEQ ID NO:The VH of the CDR of 29-31, and/or comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 32-34.Preferably, such monoclonal antibody includes, such as SEQ ID NO:VH shown in 10 and/or such as SEQ ID NO:VL shown in 12.It is highly preferred that such monoclonal antibody is monoclonal antibody 12B9.

In a preferred embodiment, the monoclonal antibody can neutralize HPV16,31 and 33 3 types HPV comprising：Including amino acid sequence is SEQ ID NO:The VH of the CDR of 35-37, and/or comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 38-40.Preferably, such monoclonal antibody includes, such as SEQ ID NO:VH shown in 14 and/or such as SEQ ID NO:VL shown in 16.It is highly preferred that such monoclonal antibody is monoclonal antibody 4H4.

On the other hand, the present invention provides the HPV that can specifically bind multiple types (for example, at least 4 types) L1 albumen and/or VLP monoclonal antibody or its antigen-binding fragment, the L1 albumen and/or VLP and choosing can be blocked From at least the 50% of the combination of following monoclonal antibody, preferably at least 60%, preferably at least 70%, preferably at least 80% are excellent Choosing at least 90%, preferably at least 95% or preferably at least 99%：

(1) monoclonal antibody generated by hybridoma cell strain 4B3, the hybridoma cell strain 4B3 are preserved in Chinese allusion quotation Type culture collection (CCTCC), and there is preserving number CCTCC-C201264；

(2) monoclonal antibody generated by hybridoma cell strain 4H4, the hybridoma cell strain 4H4 are preserved in Chinese allusion quotation Type culture collection (CCTCC), and there is preserving number CCTCC-C201265；

(3) monoclonal antibody generated by hybridoma cell strain 12B9, the hybridoma cell strain 12B9 are preserved in China Type Tissue Collection (CCTCC), and there is preserving number CCTCC-C201266；With

(4) monoclonal antibody generated by hybridoma cell strain 13A10, during the hybridoma cell strain 13A10 is preserved in State's Type Tissue Collection (CCTCC), and there is preserving number CCTCC-C201267.

The epitope that such monoclonal antibody is identified is identical as the epitope that monoclonal antibody 4B3,13A10,12B9 or 4H4 are identified, or in sky Between it is upper there is overlapping, to which such monoclonal antibody can reduce the L1 albumen (or VLP) of monoclonal antibody 4B3,13A10,12B9 or 4H4 and HPV Combination at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% or preferably at least 99%.

Conventional method such as Antibodies may be used:A Laboratory Manual,Cold Spring Harbor Method described in Laboratory, Ed Harlow and David Lane (1988), measuring a certain monoclonal antibody to be measured reduces certain The ability of monoclonal antibody combination HPV VLP known to one.One illustrative method includes：It is first that antigen is pre-coated on microwell plate, so Above-mentioned pre- packet is added in the labeled known monoclonal antibody of the unlabelled test antibodies and certain concentration be serially diluted jointly afterwards It is incubated in microwell plate by after, is then measured after washing under the test antibodies of different dilutions, it is known that antibody combines Quantity on to plate.The ability that test antibodies compete known antibodies combination antigen is stronger, it is known that the ability of antibodies bind antigen is just Weaker, the known antibodies being attached on plate are fewer.In general, antigen is pre-coated on 96 hole microwell plates, and utilize radiation mark Notation or enzyme labelling method measure the ability of the labeled known monoclonal antibody of MAbs blocking to be measured.

Kohler etc. may be used in Nature 256:The hybridoma preparation method reported in 495 (1975) prepares list It is anti-.First with immunogene (adding adjuvant when necessary) inoculation mouse or other suitable host animals.Immunogene or assistant The injection system of agent is usually subcutaneous multi-point injection or intraperitoneal injection.Immunogene is pre-conjugated certain known albumen (such as blood Pure albumen) on, it might have the immunogenicity for helping enhancement antigen in host.Adjuvant can utilize Freund's adjuvant or MPL- TDM etc..Animal will produce the lymphocyte of the antibody of secretion specific binding immunogene in vivo after receiving to be immunized.Collect mesh Lymphocyte, be used in combination suitable fusion agent (such as PEG) to merge it with myeloma cell, to obtain hybridoma (Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,Academic Press,1996)。

The hybridoma of above-mentioned preparation is inoculated into suitable culture medium and is grown, contains one in the culture medium Kind or a variety of substances that can inhibit parental myeloma cells growth that is not merging.For example, for lacking enzyme hypoxanthine guanine The parental myeloma cells of phosphotransferase (HGPRT or HPRT) add hypoxanthine, aminopterin-induced syndrome and thymus gland in the medium The substances such as pyrimidine (HAT culture mediums) will can inhibit the growth of HGPRT- deficient cells.

Preferred myeloma cell should have fusion rate high, and antibody-secreting ability is stablized, sensitive to HAT culture mediums to wait energy Power.Wherein, myeloma cell's first choice mouse source myeloma, as MOP-21 and MC-11 mouse tumors derive strain (THE Salk Institute Cell Distribution Center, San Diego, Calif.USA) and SP-2/0 or X63-Ag8- 653 cell strains (American Type C μ lture Collection, Rockville, Md.USA).Furthermore it is also possible to utilize Human myeloma and people's mouse allogenic bone marrow tumor cell strain prepare people's monoclonal antibody (Kozbor, J.Immunol., 133:3001(1984)； Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp.51-63,Marcel Dekker,Inc.,New York,1987)。

The culture medium of Growth of Hybridoma Cell is used to detect the generation of the monoclonal antibody for specific antigen.Following side can be used Method come measure hybridoma generation monoclonal antibody binding specificity：Experiment is immunoprecipitated or combines in vitro, as radio-immunity tries Test (RIA), enzyme-linked immunosorbent assay (ELISA).For example, using Munson etc. in Anal.Biochem.107:220(1980) Described in Scatchard analytic approach can measure the affinity of monoclonal antibody.

After determining the specificity of antibody of hybridoma generation, affinity and reactivity, aim cell strain can pass through Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,Academic The limiting dilution assay of Press, 1996 descriptions carry out subcloning.Suitable culture medium can be DMEM or RPMI-1640 etc..Separately Outside, hybridoma can in animal body be grown in the form of ascites tumor.

Using traditional immunoglobulin purification method, such as protein A agarose gel, hydroxyapatite chromatography, gel electricity Swimming, dialysis or affinity chromatography etc. can isolate the monoclonal antibody that subcloned cells are secreted from cell culture fluid, ascites or serum Come.

Monoclonal antibody can also be obtained by genetic engineering recombinant technique.Utilize specific binding monoclonal antibody heavy chain and light chain The nucleic acid primer of gene carries out PCR amplification, can from hybridoma isolated coding monoclonal antibody heavy chain and light chain gene DNA molecular.The DNA molecular of gained is inserted into expression vector, then transfection host cell (such as E.coli cells, COS cells, Chinese hamster ovary celI or other myeloma cells for not generating immunoglobulin), and cultivated under suitable conditions, it can obtain The target antibody of recombinant expression.

The present invention also provides the nucleic acid molecules of separation, encode the monoclonal antibody or its antigen binding fragment of the present invention Section.Such nucleic acid molecules can be isolated from hybridoma, and genetic engineering recombinant technique or chemistry can also be utilized to close It is obtained at method.

In one aspect, the present invention provides the nucleic acid molecules of separation, and it includes be capable of encoding antibody heavy variable region Nucleic acid sequence, wherein the heavy chain of antibody variable region includes：

(1) amino acid sequence is SEQ ID NO:The CDR of 17-19；

(2) amino acid sequence is SEQ ID NO:The CDR of 23-25；

(3) amino acid sequence is SEQ ID NO:The CDR of 29-31；Or

(4) amino acid sequence is SEQ ID NO:The CDR of 35-37.

In a preferred embodiment, the heavy chain of antibody variable region has SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10 or SEQ ID NO:Amino acid sequence shown in 14.

In a preferred embodiment, the nucleic acid molecules have SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9 or SEQ ID NO:Nucleotide sequence shown in 13.

On the other hand, the present invention provides the nucleic acid molecules of separation, and it includes being capable of encoding antibody light variable region Nucleic acid sequence, wherein the antibody light chain variable region includes：

(1) amino acid sequence is SEQ ID NO:The CDR of 20-22；

(2) amino acid sequence is SEQ ID NO:The CDR of 26-28；

(3) amino acid sequence is SEQ ID NO:The CDR of 32-34；With

(4) amino acid sequence is SEQ ID NO:The CDR of 38-40.

In a preferred embodiment, the antibody light chain variable region has SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:Amino acid sequence shown in 16.

In a preferred embodiment, the nucleic acid molecules have SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11 or SEQ ID NO:Nucleotide sequence shown in 15.

On the other hand, the present invention provides a kind of carriers, and it includes the nucleic acid molecules of the separation of the present invention.The present invention Carrier can be cloning vector, can also be expression vector.

In a preferred embodiment, carrier of the invention is such as plasmid, clay, bacteriophage, coemid etc..

On the other hand, the nucleic acid molecules of the separation comprising the present invention or the host cell of carrier are additionally provided.It is such Host cell includes but not limited to prokaryotic cell such as Bacillus coli cells and eukaryocyte such as yeast cells, and insect is thin Born of the same parents, plant cell and zooblast (such as mammalian cell, such as mouse cell, people's cell etc.).The cell of the present invention may be used also To be cell line, such as 293T cells.

On the other hand, the method for additionally providing the monoclonal antibody or its antigen-binding fragment that prepare the present invention, Including cultivating the host cell of the present invention under suitable conditions, and recycle monoclonal of the invention from cell culture and resist Body or its antigen-binding fragment.

On the other hand, the present invention provides selected from following hybridoma cell strain：

(1) hybridoma cell strain 4B3 is preserved in China typical culture collection center (CCTCC), and has preserving number CCTCC-C201264；

(2) hybridoma cell strain 4H4 is preserved in China typical culture collection center (CCTCC), and has preserving number CCTCC-C201265；

(3) hybridoma cell strain 12B9 is preserved in China typical culture collection center (CCTCC), and has preservation Number CCTCC-C201266；With

(4) hybridoma cell strain 13A10 is preserved in China typical culture collection center (CCTCC), and has preservation Number CCTCC-C201267.

As the application is confirmed, the amino acid sequence such as SEQ ID NO of the heavy chain variable region of monoclonal antibody 4B3:2 institutes Show (its Exemplary nucleotide sequences such as SEQ ID NO:Shown in 1), and the amino acid sequence of light chain variable region such as SEQ ID NO:4 Shown (its Exemplary nucleotide sequences such as SEQ ID NO:Shown in 3).

The amino acid sequence of CDR1, CDR2 and CDR3 of monoclonal antibody 4B3 heavy chains are respectively SEQ ID NO:17-19； The amino acid sequence of CDR1, CDR2 and CDR3 of light chain are respectively SEQ ID NO:20-22.

As the application is confirmed, the amino acid sequence such as SEQ ID NO of the heavy chain variable region of monoclonal antibody 13A10:6 Shown (its Exemplary nucleotide sequences such as SEQ ID NO:Shown in 5), and the amino acid sequence of light chain variable region such as SEQ ID NO:(its Exemplary nucleotide sequences such as SEQ ID NO shown in 8:Shown in 7).

The amino acid sequence of CDR1, CDR2 and CDR3 of monoclonal antibody 13A10 heavy chains are respectively SEQ ID NO:23- 25；The amino acid sequence of CDR1, CDR2 and CDR3 of light chain are respectively SEQ ID NO:26-28.

As the application is confirmed, the amino acid sequence such as SEQ ID NO of the heavy chain variable region of monoclonal antibody 12B9:10 Shown (its Exemplary nucleotide sequences such as SEQ ID NO:Shown in 9), and the amino acid sequence of light chain variable region such as SEQ ID NO:(its Exemplary nucleotide sequences such as SEQ ID NO shown in 12:Shown in 11).

The amino acid sequence of CDR1, CDR2 and CDR3 of monoclonal antibody 12B9 heavy chains are respectively SEQ ID NO:29-31； The amino acid sequence of CDR1, CDR2 and CDR3 of light chain are respectively SEQ ID Nos:32-34.

As the application is confirmed, the amino acid sequence such as SEQ ID NO of the heavy chain variable region of monoclonal antibody 4H4:14 Shown (its Exemplary nucleotide sequences such as SEQ ID NO:Shown in 13), and the amino acid sequence of light chain variable region such as SEQ ID NO:(its Exemplary nucleotide sequences such as SEQ ID NO shown in 16:Shown in 15).

The amino acid sequence of CDR1, CDR2 and CDR3 of monoclonal antibody 4H4 heavy chains are respectively SEQ ID NO:35-37； The amino acid sequence of CDR1, CDR2 and CDR3 of light chain are respectively SEQ ID NO:38-40.

On the other hand, the present invention provides a kind of kits comprising monoclonal antibody of the invention or its antigen Binding fragment.In a preferred embodiment, monoclonal antibody of the invention or its antigen-binding fragment further include that can examine The label of survey.In a preferred embodiment, the kit further includes secondary antibody, the specific recognition present invention's Monoclonal antibody or its antigen-binding fragment.Preferably, the secondary antibody further includes detectable label.It is such detectable It is well known to those skilled in the art when label, including but not limited to, radioactive isotope, fluorescent material, luminescent substance, colored substance Matter and enzyme (such as horseradish peroxidase) etc..

On the other hand, the present invention provides the presence in the sample of detection HPV L1 albumen or VLP or it is horizontal Method comprising use the monoclonal antibody or its antigen-binding fragment of the present invention.In a preferred embodiment, this hair Bright monoclonal antibody or its antigen-binding fragment further include detectable label.In another preferred embodiment, institute The method of stating further includes that the monoclonal antibody or its antigen knot of the present invention are detected using the secondary antibody for carrying detectable label Close segment.The method can be used for diagnostic purpose or non-diagnostic purpose (for example, the sample is cell sample, rather than is come From the sample of patient).

On the other hand, the present invention provides the methods whether diagnosis subject has infected HPV comprising：Use this The presence of the monoclonal antibody of invention or its antigen-binding fragment detection HPV L1 albumen in the sample from the subject. In a preferred embodiment, monoclonal antibody of the invention or its antigen-binding fragment further include detectable label. In another preferred embodiment, the method further includes being detected using the secondary antibody for carrying detectable label The monoclonal antibody or its antigen-binding fragment of the present invention.

On the other hand, monoclonal antibody of the invention or its antigen-binding fragment are provided in reagent preparation box Purposes, the kit is used to detect HPV L1 albumen or VLP presence in the sample or it is horizontal, or for diagnosing subject Whether HPV has been infected.

In a preferred embodiment, the monoclonal antibody is such antibody comprising：Including amino acid sequence It is classified as SEQ ID NO:The VH of the CDR of 17-19, and/or comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 20-22；It is excellent Selection of land comprising：Such as SEQ ID NO:VH shown in 2 and/or such as SEQ ID NO:VL shown in 4；It is highly preferred that it is monoclonal antibody 4B3, and the HPV be selected from HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52, HPV58 and HPV59。

In a preferred embodiment, the monoclonal antibody is such antibody comprising：Including amino acid sequence It is classified as SEQ ID NO:The VH of the CDR of 23-25, and/or comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 26-28；It is excellent Selection of land comprising：Such as SEQ ID NO:VH shown in 6 and/or such as SEQ ID NO:VL shown in 8；It is highly preferred that it is monoclonal antibody 13A10, and the HPV is selected from HPV16, HPV31, HPV33, HPV35, HPV52 and HPV58.

In a preferred embodiment, the monoclonal antibody is such antibody comprising：Including amino acid sequence It is classified as SEQ ID NO:The VH of the CDR of 29-31, and/or comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 32-34；It is excellent Selection of land comprising：Such as SEQ ID NO:VH shown in 10 and/or such as SEQ ID NO:VL shown in 12；It is highly preferred that it is single Anti- 12B9, and the HPV is selected from HPV16, HPV31, HPV33, HPV35, HPV52 and HPV58.

In a preferred embodiment, the monoclonal antibody is such antibody comprising：Including amino acid sequence It is classified as SEQ ID NO:The VH of the CDR of 35-37, and/or comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 38-40；It is excellent Selection of land comprising：Such as SEQ ID NO:VH shown in 14 and/or such as SEQ ID NO:VL shown in 16；It is highly preferred that it is single Anti- 4H4, and the HPV is selected from HPV16, HPV31, HPV33, HPV35 and HPV52.

On the other hand, the present invention provides a kind of pharmaceutical composition, it includes the monoclonal antibody of the present invention or its Antigen-binding fragment and pharmaceutically acceptable carrier and/or excipient.In a preferred embodiment, the list Clonal antibody is selected from following：

(1) monoclonal antibody comprising：Including amino acid sequence is SEQ ID NO:The VH of the CDR of 23-25, and/or packet It is SEQ ID NO containing amino acid sequence:The VL of the CDR of 26-28；Preferably comprising：Such as SEQ ID NO:VH shown in 6 and/ Or such as SEQ ID NO:VL shown in 8；It is highly preferred that it is monoclonal antibody 13A10；

(2) monoclonal antibody comprising：Including amino acid sequence is SEQ ID NO:The VH of the CDR of 29-31, and/or packet It is SEQ ID NO containing amino acid sequence:The VL of the CDR of 32-34；Preferably comprising：Such as SEQ ID NO:VH shown in 10 And/or such as SEQ ID NO:VL shown in 12；It is highly preferred that it is monoclonal antibody 12B9；Or

(3) monoclonal antibody comprising：Including amino acid sequence is SEQ ID NO:The VH of the CDR of 35-37, and/or packet It is SEQ ID NO containing amino acid sequence:The VL of the CDR of 38-40；Preferably comprising：Such as SEQ ID NO:VH shown in 14 And/or such as SEQ ID NO:VL shown in 16；It is highly preferred that it is monoclonal antibody 4H4.

On the other hand, the present invention provides HPV infections for preventing or treating subject or related to HPV infection Disease (such as cervical carcinoma) method comprising, to subject in need application prevent or therapeutically effective amount this hair Bright pharmaceutical composition.

On the other hand, the monoclonal antibody or its antigen-binding fragment for providing the present invention are preparing pharmaceutical composition In purposes, described pharmaceutical composition be used for prevent or treat subject HPV infection or with the relevant disease (example of HPV infection Such as cervical carcinoma).

In a preferred embodiment, the monoclonal antibody is such antibody comprising：Including amino acid sequence It is classified as SEQ ID NO:The VH of the CDR of 23-25, and/or comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 26-28；It is excellent Selection of land comprising：Such as SEQ ID NO:VH shown in 6 and/or such as SEQ ID NO:VL shown in 8；It is highly preferred that it is monoclonal antibody 13A10；And the HPV is selected from HPV16,31,33 and 58.

In a preferred embodiment, the monoclonal antibody is such antibody comprising：Including amino acid sequence It is classified as SEQ ID NO:The VH of the CDR of 29-31, and/or comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 32-34；It is excellent Selection of land comprising：Such as SEQ ID NO:VH shown in 10 and/or such as SEQ ID NO:VL shown in 12；It is highly preferred that it is single Anti- 12B9；And the HPV is selected from HPV33 and 58.

In a preferred embodiment, the monoclonal antibody is such antibody comprising：Including amino acid sequence It is classified as SEQ ID NO:The VH of the CDR of 35-37, and/or comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 38-40；It is excellent Selection of land comprising：Such as SEQ ID NO:VH shown in 14 and/or such as SEQ ID NO:VL shown in 16；It is highly preferred that it is single Anti- 4H4；And the HPV is selected from HPV16,31 and 33.

On the other hand, the present invention provides the methods for neutralizing the virulence of HPV in sample comprising, will include The sample of HPV is contacted with the monoclonal antibody of the present invention or its antigen-binding fragment.Such method can be used for therapeutic purposes, or Non-treatment purpose (such as the sample is cell sample, rather than patient or the sample from patient).On the other hand, it carries Monoclonal antibody of the invention or its antigen-binding fragment has been supplied to be used to prepare the purposes of drug, the drug is for neutralizing sample The virulence of middle HPV.

In a preferred embodiment, the HPV is selected from HPV16,31,33 and 58, and the monoclonal antibody is Such antibody comprising：Including amino acid sequence is SEQ ID NO:The VH of the CDR of 23-25, and/or include amino acid sequence It is classified as SEQ ID NO:The VL of the CDR of 26-28；Preferably comprising：Such as SEQ ID NO:VH shown in 6 and/or such as SEQ ID NO:VL shown in 8；It is highly preferred that it is monoclonal antibody 13A10.

In a preferred embodiment, the HPV is selected from HPV33 and 58, and the monoclonal antibody is such Antibody comprising：Including amino acid sequence is SEQ ID NO:The VH of the CDR of 29-31, and/or comprising amino acid sequence be SEQ ID NO:The VL of the CDR of 32-34；Preferably comprising：Such as SEQ ID NO:VH shown in 10 and/or such as SEQ ID NO:12 Shown in VL；It is highly preferred that it is monoclonal antibody 12B9.

In a preferred embodiment, the HPV is selected from HPV16,31 and 33, and the monoclonal antibody is in this way Antibody comprising：Including amino acid sequence is SEQ ID NO:The VH of the CDR of 35-37, and/or be comprising amino acid sequence SEQ ID NO:The VL of the CDR of 38-40；Preferably comprising：Such as SEQ ID NO:VH shown in 14 and/or such as SEQ ID NO:VL shown in 16；It is highly preferred that it is monoclonal antibody 4H4.

On the other hand, the present invention also provides a kind of epitope peptides of separation, are connected by 6-15 of HPV L1 albumen Continuous amino acid residue composition, and include the 303-308 amino acids residues of HPV L1 albumen.In a preferred embodiment party In case, the HPV L1 albumen is HPV16L1 albumen.In a preferred embodiment, the of the HPV L1 albumen 303-308 amino acids residue such as SEQ ID NO:Shown in 58.

In a preferred embodiment, epitope peptide of the invention by L1 albumen not more than 15 continuous amino acids Residue forms, for example, it is residual by 15,14,13,12,11,10,9,8,7 or 6 continuous amino acids Base forms.For example, the epitope peptide of the present invention, which has, is selected from SEQ ID NO:Amino acid sequence shown in 41-43 and 50-56.

Particularly, epitope peptide of the invention, does not need carrier protein, can be known by monoclonal antibody 4B3 specificity Not/combine.It is alternatively possible to the epitope peptide of the present invention be merged with carrier protein, to promote presentation and the enhancing epitope of epitope The immunogenicity of peptide.Therefore, the present invention also provides a kind of recombinant proteins, and it includes the epitope peptides and carrier of the separation of the present invention Albumen, and not naturally occurring albumen or its segment.In the recombinant protein, the epitope peptide can be connected to carrier The N-terminal or C-terminal of albumen are inserted into the inside of carrier protein, or replace the partial amino-acid series of carrier protein, depending on being used Specific carrier protein depending on.Preferably, the carrier protein is selected from HbcAg and CRM197 albumen.In addition, optionally Ground, the epitope peptide pass through connector (rigidity or flexible connection body, such as (GGGGS)₃) be connected with carrier protein.The present invention Recombinant protein do not limited by its producing method, for example, it can be generated by gene engineering method (recombinant technique), also may be used To be generated by chemical synthesis process.

On the other hand, the present invention also provides a kind of nucleic acid molecules of separation, and it includes the epitopes of the coding present invention The nucleotide sequence of peptide or recombinant protein.On the other hand, the present invention also provides a kind of carrier, it includes as described above The nucleic acid molecules of separation.The carrier of the present invention can be cloning vector, can also be expression vector.In a preferred embodiment In, carrier of the invention is such as plasmid, clay, bacteriophage, coemid etc..

On the other hand, the host cell for including the nucleic acid molecules or carrier detached as described above is additionally provided.This Class host cell includes but not limited to prokaryotic cell such as Bacillus coli cells and eukaryocyte such as yeast cells, insect Cell, plant cell and zooblast (such as mammalian cell, such as mouse cell, people's cell etc.).The cell of the present invention is also Can be cell line, such as 293T cells.

On the other hand, the method for preparing the recombinant protein of the present invention is additionally provided comprising, under suitable conditions Host cell as described above is cultivated, and recycles the recombinant protein of the present invention from cell culture.

Advantageous effect of the invention

Compared with prior art, monoclonal antibody of the invention and its antigen-binding fragment have significant advantageous aspect. Particularly, monoclonal antibody of the invention and its antigen-binding fragment can identify to wide spectrum/combine a variety of types (it is at least five kinds of, Even up to 11 kinds) the L1 albumen and VLP of HPV, to which it has especially significant advantage in terms of detect and diagnose.

In addition, the present invention also provides monoclonal antibodies and its antigen-binding fragment with wide spectrum neutralization activity, for example, Monoclonal antibody 13A10 can neutralize the HPV of HPV16,31,33 and 58 4 types；Monoclonal antibody 12B9 can be neutralized The HPV of HPV33 and 58 two type；Monoclonal antibody 4H4 can neutralize the HPV of HPV16,31 and 33 3 types.This for Prevent or treat the HPV infection of subject or there is especially significant advantage with the relevant disease of HPV infection (such as cervical carcinoma).

Embodiment of the present invention is described in detail below in conjunction with drawings and examples, but people in the art Member will be understood that following drawings and embodiment are merely to illustrate the present invention, rather than to the restriction of the scope of the present invention.With reference to the accompanying drawings With the following detailed description of preferred embodiment, various purposes of the invention and advantageous aspect are to those skilled in the art It will be apparent.

Description of the drawings

Fig. 1 shows the testing result of western blot analysis, and which show the other HPV L1 albumen of different shaped and 4B3 Specific binding.Wherein, protein sample used in each swimming lane is as follows：Swimming lane 1：HPV6L1 albumen；Swimming lane 2：HPV11L1 eggs In vain；Swimming lane 3：HPV16L1 albumen；Swimming lane 4：HPV18L1 albumen；Swimming lane 5：HPV31L1 albumen；Swimming lane 6：HPV33L1 albumen；Swimming Road 7：EV71 albumen；Swimming lane 8：CRM197 albumen；Swimming lane 9：HEV495 albumen；Swimming lane 10：HPV35L1 albumen；Swimming lane 11： HPV45L1 albumen；Swimming lane 12：HPV52L1 albumen；Swimming lane 13：Humanpapilloma virus 58 L1 protein；Swimming lane 14：HPV59L1 albumen；Swimming lane 15： EV71 albumen；Swimming lane 16：CRM197 albumen；Swimming lane 17：HEV495 albumen.Wherein, a concentration of 5 μ of the protein sample of each swimming lane G/ml, and a concentration of 10 μ g/ml of used antibody 4B3.The results show that the HPV L1 albumen for the 11 kinds of types tested is equal Specific reaction can occur with 4B3, and three kinds of unrelated proteins cannot be reacted with 4B3.This illustrates that monoclonal antibody 4B3 can wide spectrum and special Property combine a variety of types HPV L1 albumen.

Fig. 2 shows the testing result of western blot analysis, and 10 which show HPV16L1 albumen are subcloned peptide (HPV16L1A-HPV16L1J) and the combination situation of monoclonal antibody 4B3.Wherein, protein sample used in each swimming lane is as follows：Swimming lane 1： HPV16L1A albumen；Swimming lane 2：HPV16L1B albumen；Swimming lane 3：HPV16L1C albumen；Swimming lane 4：HPV16L1D albumen；Swimming lane 5： HPV16L1E albumen；Swimming lane 6：HPV16L1F albumen；Swimming lane 7：HPV16L1G albumen；Swimming lane 8：HPV16L1H albumen；Swimming lane 9： HPV16L1I albumen；Swimming lane 10：HPV16L1J albumen；Swimming lane M：Protein markers).The results show that HPV16L1 albumen Peptide fragment HPV16L1F can be reacted with HPV16L1G with 4B3, and remaining peptide fragment cannot be reacted with 4B3.This shows monoclonal antibody 4B3 institutes In conjunction with epitope be located at position Chong Die with HPV16L1G peptide fragment HPV16L1F (small peptide of fifteen amino acid, SEQ ID NO: 41)。

Fig. 3 shows the testing result of western blot analysis, and which show the other HPVL1 albumen of different shaped and difference are dense The combination situation of the monoclonal antibody 4B3 of degree.Wherein, protein sample used in each swimming lane (a concentration of 5 μ g/ml) is as follows：

Swimming lane 1,8,15,22:HPV6L1 albumen；

Swimming lane 2,9,16,23:HPV11L1 albumen；

Swimming lane 3,10,17,24:HPV16L1 albumen；

Swimming lane 4,11,18,25:HPV18L1 albumen；

Swimming lane 5,12,19,26:HPV33L1 albumen；

Swimming lane 6,13,20,27:HPV52L1 albumen；

Swimming lane 7,14,21,28:Humanpapilloma virus 58 L1 protein.

Wherein,

Swimming lane 1-7 is detected using the 4B3 of 10 μ g/ml；

Swimming lane 8-14 is detected using the 4B3 of 1 μ g/ml；

Swimming lane 15-21 is detected using the 4B3 of 0.1 μ g/ml；

Swimming lane 22-28 is detected using the 4B3 of 0.01 μ g/ml.

The results show that as a concentration of at least 0.1 μ g/ml of the monoclonal antibody 4B3 used, it is able to detect that 11 kinds of types HPV L1 albumen.

Fig. 4 shows the testing result of western blot analysis, and which show the HPV L1 of the various types of various concentration The combination situation of the monoclonal antibody 4B3 of albumen and 1 μ g/ml.Wherein, protein sample used in each swimming lane is as follows：

Swimming lane 1-7:HPV6L1 albumen, concentration respectively are 100 μ g/ml, 33 μ g/ml, 11 μ g/ml, 3.6 μ g/ml, 1.2 μ g/ml, 0.4 μ g/ml, 0.1 μ g/ml；

Swimming lane 8-14:HPV11L1 albumen, concentration respectively are 100 μ g/ml, 33 μ g/ml, 11 μ g/ml, 3.6 μ g/ml, 1.2 μ g/ml, 0.4 μ g/ml, 0.1 μ g/ml；

Swimming lane 15-21:HPV16L1 albumen, concentration respectively are 100 μ g/ml, 33 μ g/ml, 11 μ g/ml, 3.6 μ g/ Ml, 1.2 μ g/ml, 0.4 μ g/ml, 0.1 μ g/ml；

Swimming lane 22-28:HPV18L1 albumen, concentration respectively are 100 μ g/ml, 33 μ g/ml, 11 μ g/ml, 3.6 μ g/ Ml, 1.2 μ g/ml, 0.4 μ g/ml, 0.1 μ g/ml；

Swimming lane 29-35:HPV31L1 albumen, concentration respectively are 100 μ g/ml, 33 μ g/ml, 11 μ g/ml, 3.6 μ g/ Ml, 1.2 μ g/ml, 0.4 μ g/ml, 0.1 μ g/ml；

Swimming lane 36-42:HPV33L1 albumen, concentration respectively are 100 μ g/ml, 33 μ g/ml, 11 μ g/ml, 3.6 μ g/ Ml, 1.2 μ g/ml, 0.4 μ g/ml, 0.1 μ g/ml；

Swimming lane 43-49:HPV45L1 albumen, concentration respectively are 100 μ g/ml, 33 μ g/ml, 11 μ g/ml, 3.6 μ g/ Ml, 1.2 μ g/ml, 0.4 μ g/ml, 0.1 μ g/ml；

Swimming lane 50-56:HPV52L1 albumen, concentration respectively are 100 μ g/ml, 33 μ g/ml, 11 μ g/ml, 3.6 μ g/ Ml, 1.2 μ g/ml, 0.4 μ g/ml, 0.1 μ g/ml；

Swimming lane 57-63:Humanpapilloma virus 58 L1 protein, concentration respectively are 100 μ g/ml, 33 μ g/ml, 11 μ g/ml, 3.6 μ g/ Ml, 1.2 μ g/ml, 0.4 μ g/ml, 0.1 μ g/ml.

The results show that the combination of the HPV L1 albumen and monoclonal antibody 4B3 for each type tested is dose-dependent；And And when carrying out WB detections using 4B3, the detectable limit of the other L1 albumen of different shaped has differences, and in 0.4 μ g/ml-3.6 μ Between g/ml.

Fig. 5, which is shown, uses monoclonal antibody 4B3, and by immunoblotting, detection 65 has different degrees of cervical lesions The analysis result of the cervical exfoliated cell sample of patient.The results show that monoclonal antibody 4B3 can be with the HPV L1 eggs in tissue of patient sample It reacts in vain, and higher to the recall rate of the HPV L1 albumen in patient samples, this is mainly reacted by the wide spectrum of monoclonal antibody 4B3 Caused by property.

Sequence information

In the table 1 that the information of sequence of the present invention is provided below.

Sequence information

(the SEQ ID NO of sequence 1:1)：471bp

ATGGACAGACTTACATCTTCATTCCTGCTGCTGATTGTCCCTGCATATGTCCTTTCCCAGGTTACTCTG AAAGAGTCTGGCCCTGGGATATTGCAGACCTCCCAGACCCTCAGTCTGACTTGTTCTTCCTCTGGGTTTTCACTGAA CACCTCTGGTATGGGTGTGAGCTGGATTCGTCAGCCTTCAGGACAGGGTCTGGAGTGGCTGGCACACACTTACTGGG ATGATGACAAGCGCTATAATCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGAAACCTCCAGAAACCAGGTACTT CTCAAGATCACCAGTGTTGACACTGCAGATACTGCCACATACTACTGTGCTCGATATTTCTACGATAGTAACTACGG AGGGATGGACTACTGGGGTCAAGGAACCTCTGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCGTCTATCCACT GGTCCCTGGAATCTCTA

(the SEQ ID NO of sequence 2:2)：157aa

MDRLTSSFLLLIVPAYVLSQVTLKESGPGILQTSQTLSLTCSSSGFSLNTSGMGVSWIRQPSGQGLEWL AHTYWDDDKRYNPSLKSRLTISKETSRNQVLLKITSVDTADTATYYCARYFYDSNYGGMDYWGQGTSVTVSSAKTTP PSSIHWSLESL

(the SEQ ID NO of sequence 3:3)：387bp

ATGGTGTCCACAGCTCAGTTCCTTGGGATCTTGTTGCTCTGGTTTCCAGGTATCAGATGTGACATCACG ATGACCCAGTCTCCATCCTCCATGTATGCATCGCTGGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACAT TAAAAGTTATTTAAGCTGGTACCAACAGAAACCAAGGAAATCTCCTAAGACCCTGATCTATTATGCAACAAGGTTGT CAGATGGGGTCCCATCAAGATTCAGTGGCAGTGGATCTGGCCAAGATTATTCTCTAACCATCAGCAGCCTGGAGTCT GACGATACAGCAACTTATTACTGTCTACAACATTATGAGAGCCCGCTCACGTTCGGTCCTGGGACCAAGCTGGAAAT AAAACGGATC

(the SEQ ID NO of sequence 4:4)：129aa

MVSTAQFLGILLLWFPGIRCDITMTQSPSSMYASLGERVTITCKASQDIKSYLSWYQQKPRKSPKTLIY YATRLSDGVPSRFSGSGSGQDYSLTISSLESDDTATYYCLQHYESPLTFGPGTKLEIKRI

(the SEQ ID NO of sequence 5:5)：426bp

ATGAAATGCAGCTGGGTTATGTTCTTCCTGATGGCAGTGGTTACAGGGGTCAATTCAGAGGTTCAGCTG CAGCAGTCTGGGGCAGACCTTGTGAAGCCAGGGGCCTCAGTCAAGCTGTCCTGCACAGCTTCTGGCTTCAACATTAG AGACACCTTTATGCAGTGGGTGAAGCAGAGGCCTGAACAGGGCCCGGAGTACATTGGAAGGATTGATCCTGCAAATG GCTATGTTGAATATGGCCCGAAGTTCCAGGACAAGGCCACAATAACGGCAGACAAATCCTCCAACACAGCCTACCTT CAACTCAGCAGCCTGACATCTGAGGACACTGCCGTCTACTTCTGTAGTTCCTTCCATGGTCAACCTTTTGCTTATTG GGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCA

(the SEQ ID NO of sequence 6:6)：142aa

MKCSWVMFFLMAVVTGVNSEVQLQQSGADLVKPGASVKLSCTASGFNIRDTFMQWVKQRPEQGPEYIGR IDPANGYVEYGPKFQDKATITADKSSNTAYLQLSSLTSEDTAVYFCSSFHGQPFAYWGQGTLVTVSAAKTTPP

(the SEQ ID NO of sequence 7:7)：417bp

ATGGGCTTCAAGATGAAGTCACAGTTTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTATTAAA GGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTGGGAGACAGGGTCAACATCACCTGCATGGC CAGTCAGGATGTGGGTTCTGCTGTTGCCTGGTATCAACAGAAACCCGGACAATCTCCTAAATTACTGATTTATTGGG CATCCTCCCGGCACATTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAAC AATGTGCAGTCTGAAGACTTGGCAGATTATTTCTGTCAACAATATAGCACCTATACGTTCGGAGGGGGGACCAAGTT GGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATC

(the SEQ ID NO of sequence 8:8)：139aa

MGFKMKSQFQVFVYMLLWLSGIKGDIVMTQSHKFMSTSVGDRVNITCMASQDVGSAVAWYQQKPGQSPK LLIYWASSRHIGVPDRFTGSGSGTDFTLTINNVQSEDLADYFCQQYSTYTFGGGTKLEIKRADAAPTVSI

(the SEQ ID NO of sequence 9:9)：459bp

ATGAGAGTGCTGATTCTTTTGTGGCTGTTCACAGCCTTTCCTGGTGTCGTGTCTGATGTGGCGCTTCAG GAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCGCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAG TGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTTCATTACCAACAGTGATA CCACTAGCTACAATCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACGCATCCAAGAACCAGTTCTTCCTACAG TTGAGTTCTGTGACTACTGAGGACACAGCCACATATTATTGTGCAAGGTCCTCGGCCCCTCATTACTACGGCCTTTA CTGGGGCCAGGGGACTCTGGTCGCTGTCTCTGCAGCCAAAACGACACCCCCACCGTCTATCCACTGGTCCCTGGAAT CTCTA

(the SEQ ID NO of sequence 10:10)：153aa

MRVLILLWLFTAFPGVVSDVALQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGF ITNSDTTSYNPSLKSRISITRDASKNQFFLQLSSVTTEDTATYYCARSSAPHYYGLYWGQGTLVAVSAAKTTPPPSI HWSLESL

(the SEQ ID NO of sequence 11:11)：435bp

ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATTCTGTCCAGAGGACAA ATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGTTC AAGTGTAAATTACATGTATTGGTACCAGCAGAAGCCAGGATCTTCCCCCAGACTCCTGATTTATGACACATCCCGCC TGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCCAAATGGAG GCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGA AATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAATCTC

(the SEQ ID NO of sequence 12:12)：145aa

MDFQVQIFSFLLISASVILSRGQIVLTQSPAIMSASPGEKVTMTCSASSSVNYMYWYQQKPGSSPRLLI YDTSRLASGVPVRFSGSGSGTSYSLTISQMEAEDAATYYCQQWSSYPYTFGGGTKLEIKRADAAPTVSIFPPSSNL

(the SEQ ID NO of sequence 13:13)：459bp

ATGAAATGCACCTGGGTTATTCTCTTTTTGATAGCAACAGCAACAGGTGTCCACTCCCAGGTCCAACTG CAGCAGTCTGGGGCTGAACTGGTGAACCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCCTCTGGCTACACCTTCAC CGACTACTATATATTTTGGGTGAAGCAGAGGCCTGGACTAGGCCTTGAGTGGATTGGAGAGATTAATCCTAACACTG GTGGTACTACCTTCAATGAGAGGTTCAAGGGCAAGGCCACACTTACTGTAGACAAATCCTCCAGTACAACATACATC CAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTACATCCTTTTACTACGGTAGTCCCTTTGACCA CTGGGGCCAAGGCACCACTCTCACAGTCTCCTCTGCCAAAACGACACCCCCATCGTTTATCCCTTGGCCCCTGGAAT CTCTA

(the SEQ ID NO of sequence 14:14)：153aa

MKCTWVILFLIATATGVHSQVQLQQSGAELVNPGASVKLSCKASGYTFTDYYIFWVKQRPGLGLEWIGE INPNTGGTTFNERFKGKATLTVDKSSSTTYIQLSSLTSEDSAVYYCTSFYYGSPFDHWGQGTTLTVSSAKTTPPSFI PWPLESL

(the SEQ ID NO of sequence 15:15)：435bp

ATGGATTTTCAAGTGCAGATTATCAGCTTCCTGCTAATCAGTGCCTCAGTCATGCTGTCCAGAGGACAA GTTGTTCTCATCCAGTCTCCAGCAATCATGTCTGTATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTTCCAGTTC AAGTATAGATTACATTCACTGGTACCAGCAGAAGCCAGGCACCTCCCCCAACAGATGGATTTATGACACATCCAAAT TGCCTTCTGGAGTCCCTGCTCGCTTCAGTAGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGTAGCATGGAG GCTGAGGATGCTGCCACTTATTACTGCCATCAGCGGAATAATTACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGA GCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAATCTC

(the SEQ ID NO of sequence 16:16)：145aa

MDFQVQIISFLLISASVMLSRGQVVLIQSPAIMSVSPGEKVTMTCSSSSSIDYIHWYQQKPGTSPNRWI YDTSKLPSGVPARFSSSGSGTSYSLTISSMEAEDAATYYCHQRNNYPLTFGAGTKLELKRADAAPTVSIFPPSSNL

(the SEQ ID NO of sequence 17:17)：10aa

GFSLNTSGMG

(the SEQ ID NO of sequence 18:18)：7aa

TYWDDDK

(the SEQ ID NO of sequence 19:19)：14aa

ARYFYDSNYGGMDY

(the SEQ ID NO of sequence 20:20)：6aa

QDIKSY

(the SEQ ID NO of sequence 21:21)：3aa

YAT

(the SEQ ID NO of sequence 22:22)：9aa

LQHYESPLT

(the SEQ ID NO of sequence 23:23)：8aa

GFNIRDTF

Sequence 24 (SEQ ID NO:24)：8aa

IDPANGYV

(the SEQ ID NO of sequence 25:25)：10aa

SSFHGQPFAY

(the SEQ ID NO of sequence 26:26)：6aa

QDVGSA

(the SEQ ID NO of sequence 27:27)：3aa

WAS

(the SEQ ID NO of sequence 28:28)：7aa

QQYSTYT

(the SEQ ID NO of sequence 29:29)：9aa

GYSITSDYA

(the SEQ ID NO of sequence 30:30)：7aa

ITNSDTT

(the SEQ ID NO of sequence 31:31)：12aa

ARSSAPHYYGLY

(the SEQ ID NO of sequence 32:32)：5aa

SSVNY

(the SEQ ID NO of sequence 33:33)：3aa

DTS

(the SEQ ID NO of sequence 34:34)：9aa

QQWSSYPYT

(the SEQ ID NO of sequence 35:35)：8aa

GYTFTDYY

(the SEQ ID NO of sequence 36:36)：8aa

INPNTGGT

(the SEQ ID NO of sequence 37:37)：11aa

TSFYYGSPFDH

(the SEQ ID NO of sequence 38:38)：5aa

SSIDY

(the SEQ ID NO of sequence 39:39)：3aa

DTS

(the SEQ ID NO of sequence 40:40)：9aa

HQRNNYPLT

(the SEQ ID NO of sequence 41:41)：15aa

AQIFNKPYWLQRAQG

(the SEQ ID NO of sequence 42:42)：14aa

QIFNKPYWLQRAQG

(the SEQ ID NO of sequence 43:43)：13aa

IFNKPYWLQRAQG

(the SEQ ID NO of sequence 44:44)：12aa

FNKPYWLQRAQG

(the SEQ ID NO of sequence 45:45)：11aa

NKPYWLQRAQG

(the SEQ ID NO of sequence 46:46)：10aa

KPYWLQRAQG

(the SEQ ID NO of sequence 47:47)：9aa

PYWLQRAQG

(the SEQ ID NO of sequence 48:48)：8aa

YWLQRAQG

(the SEQ ID NO of sequence 49:49)：7aa

WLQRAQG

(the SEQ ID NO of sequence 50:50)：14aa

AQIFNKPYWLQRAQ

(the SEQ ID NO of sequence 51:51)：13aa

AQIFNKPYWLQRA

(the SEQ ID NO of sequence 52:52)：12aa

AQIFNKPYWLQR

(the SEQ ID NO of sequence 53:53)：11aa

AQIFNKPYWLQ

(the SEQ ID NO of sequence 54:54)：10aa

AQIFNKPYWL

(the SEQ ID NO of sequence 55:55)：9aa

AQIFNKPYW

(the SEQ ID NO of sequence 56:56)：8aa

AQIFNKPY

(the SEQ ID NO of sequence 57:57)：7aa

AQIFNKP

(the SEQ ID NO of sequence 58:58)：6aa

IFNKPY

(the SEQ ID NO of sequence 59:59)：502aa

MLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNNNKILVPKVSGLQYRV FRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGVGISGHPLLNKLDDTENASAYAANAGVDNRECISM DYKQTQLCLIGCKPPIGEHWGKGSPCTNVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSEVPLDICT SICKYPDYIKMVSEPYGDSLFFYLRREQMFVRHLFNRAGAVGDNVPDDLYIKGSGSTANLASSNYFPTPSGSMVTSD AQIFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMSLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCK ITLTADIMTYIHSMNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPKEDPLKKYTFWEVNLKEKFSA DLDQFPLGRKFLLQAGLEAKPKFTLGKRKATPTTSSTSTTAKRKKRKL

Explanation about biomaterial preservation

It has been carried out in China typical culture collection center (CCTCC, Wuhan, China, Wuhan University) the present invention relates to following The biomaterial of preservation：

Hybridoma cell strain 4B3, preserving number CCTCC-C201264, preservation time are on May 31st, 2012；

Hybridoma cell strain 4H4, preserving number CCTCC-C201265, preservation time are on May 31st, 2012；

Hybridoma cell strain 12B9, preserving number CCTCC-C201266, preservation time are on May 31st, 2012；

Hybridoma cell strain 13A10, preserving number CCTCC-C201267, preservation time are on May 31st, 2012.

Specific implementation mode

It is intended to illustrate the present invention embodiment (rather than limiting the invention) to describe the present invention referring now to following.

Unless specifically stated otherwise, the experimental methods of molecular biology and immunodetection used in the present invention, substantially joins According to J.Sambrook et al., molecular cloning：Laboratory manual, second edition, CSH Press, 1989, and F.M.Ausubel et al., fine works molecular biology experiment guide, the 3rd edition, described in John Wiley＆Sons, Inc., 1995 Method carry out；The condition that the use of restriction enzyme is recommended according to goods producer.The examination in source is not specified in embodiment Agent is the conventional reagent of this field or commercially available reagent.Those skilled in the art know that embodiment is retouched by way of example The present invention is stated, and is not intended to limit scope of the present invention.

The preparation and the reactive analysis of wide spectrum of 1. anti-HPVL1 protein monoclonal antibodies of embodiment

This experiment passes through the HPV VLP cross immunity mouse with multiple types, by the splenocyte and bone of immunized mouse Myeloma cells merge, and are screened, and obtain having the thin of reactive antibody and secretory antibody to the HPV VLP of multiple types Born of the same parents' strain.

The preparation of antigen

Using escherichia expression system, the L1 albumen of the type of HPV16,33,52,58 is prepared, and passes through transmission electron microscope observing It is uncommon etc. that its form (Wei Min is detected with dynamic light scattering detection method, the preparation of HPV 16 virus-like particle and its is exempted from Epidemic focus Journal of Sex Research, viral journal, 2009,4 (25)).The results show that 4 kinds of obtained L1 albumen can be formed diameter about 55nm, with The similar particle of natural viral particle shape height, that is, form can be used for being immunized mouse virus-like particle (HPV16, The VLP of HPV33, HPV52, HPV58).4 kinds of albumen obtained above is diluted to 10 μ g/ml.

Mouse

6 week old female Balb/c mouse are provided by Xiamen University's school of life and health sciences Experimental Animal Center.

The preparation of hybridoma

We prepare monoclonal antibody using the vivo immunization mode of standard and PEG fusion methods.Method detailed referring to Ed Harlow et al.,“Antibodies A Laboratory Manual”,Cold Spring Harbor Laboratory 1988.The simplified process of this method is as follows：

Mouse immune：HPV16, HPV 33 obtained above, HPV 52, HPV 58 VLP in, an optional type HPV VLP and Freund's complete adjuvant (CFA) isometric mixing and emulsifying, carry out limb muscle multi-point injection, every per injection 250 μl.14d, 28d and 42d after first immunisation add Freund not exclusively to help with the HPV VLP of same dose of in addition any type respectively Agent (IFA) carries out booster immunization.After the 3rd booster immunization, blood sampling detects it and reacts titre with HPV VLP.When titre reaches To 10⁶When above, take Mouse spleen cells for merging.72hr carries out booster immunization again before fusion：Through tail vein injection 1 Secondary, 50 μ l/ are only.Prepare 60 blocks of fusion plates.

Fusion：The spleen cell for taking highest 3 mouse of serum titer, blends with murine myeloma cell.First by spleen Dirty grinding, obtains splenocyte suspension；Then by it with cell number low ten times of SP2/0 mouse bone marrow cells in exponential phase Oncocyte mixes, and acts on 1min through PEG1500, together by two kinds of cell fusions；Then fused cell liquid (1200ml) point It is attached in 60 piece of 96 orifice plate and cultivates.Fusion culture medium is the complete screening and culturing mediums of RPMI1640 containing HAT and 20%FBS.Antigen Broad spectrum activity clone is obtained by ELISA and neutralization experiment screening, and after 3 time clonings, obtains stable monoclonal antibody Cell strain.

The screening of hybridoma：After fused cell is cultivated 10 days in 96 orifice plates, draw cell conditioned medium, carry out ELISA and in And detection.Cloning is continued into positive hole, until the antibody secreted by cell strain stable bond HPV VLP and can neutralize Until HPV pseudovirus.

The selection result：Obtain the cell strain of four plants of secrete monoclonal antibodies：4B3、13A10、12B9、4H4.

The culture of hybridoma：By stable hybridoma cell strain in carbon dioxide incubator amplification cultivation：From 96 orifice plates 24 orifice plates are transferred to, 50ml Tissue Culture Flasks are transferred to.Then, the cell in Tissue Culture Flask is collected, is injected into small Mouse is intraperitoneal, and draws ascites from mouse peritoneal after 7-10 days.

The purifying of monoclonal antibody

Ascites is first handled with 50% ammonium sulfate precipitation, is then dialysed to PBS, pH7.2, is then carried out with DEAE columns HPLC is purified, and finally obtains purified monoclonal antibody.SDS-PAGE detection displays, the purity of purified monoclonal antibody Reach 95% or more.

Monoclonal antibody and the reactive ELISA of the HPV VLP of each type are detected

By HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 45, HPV 52, HPV The VLP of this 11 types of 58 and HPV 59 is adsorbed to respectively on 96 orifice plates, is then obtained with 3 times of concentration gradient dilution additions Monoclonal antibody (maximum concentration 1mg/ml).The combination that the HPV VLP of each monoclonal antibody and each type are detected by ELISA is strong Degree.

The greatest dilution that antibody can be reacted with VLP is directed to the antibody titer of the VLP as the antibody.When antibody drips When degree is higher than 10, it is believed that the antibody can be combined with the VLP of the type.It is computed, 4B3,13A10,4H4 and 12B9 are directed to each type (result is with log as shown in table 2 for the antibody titer of other HPV VLP₁₀Antibody titer indicates).The results show that this 4 plants of monoclonal antibodies have There is wide spectrum reactive：They can react with the HPV VLP of at least five type.Wherein, 4B3 and tested 11 The HPV VLP of type react；And 13A10 can be with HPV 16, HPV 31, HPV 33, HPV 35, HPV 52, HPV 58 VLP react；4H4 can react with the VLP of HPV 16, HPV 31, HPV 33, HPV 35, HPV 52；12B9 can be with HPV 16, HPV 31, HPV 33, HPV 35, HPV 52, HPV58 VLP react.

Table 2：The response intensity of the HPV VLP of 4 kinds of monoclonal antibodies and 11 types

The reactive Western blotting (WB) of monoclonal antibody and the HPV L1 albumen of each type detects

Whether can be reacted with HPV L1 albumen with the analysis of Western blotting (WB) method 4B3,13A10,4H4 and 12B9.If energy Reaction then illustrates that the epitope that the antibody is identified is linear epitope；Conversely, then illustrating that the epitope that the antibody is identified is conformation table Position.

SDS- polyacrylamide gel electrophoresises：The L1 albumen of HPV6,11,16,18,31,33,45,52,58 is diluted to 5 μ G/ml carries out SDS- polyacrylamide gel electrophoresises.

Transferring film：It will be on the albumen electrotransfer to nitrocellulose filter on SDS- polyacrylamide gels.

Closing：Nitrocellulose filter is immersed in 5% defatted milk, 60min is closed.

Add antibody:Each antibody is diluted to 10 μ g/ml with 5% defatted milk respectively, it is then small with nitrocellulose film reaction 1 When.

Washing：With 20mM Tirs-HCl, pH8.0,0.5M NaCl, 0.05%Tween20 solution washs nitrocellulose Film 5 times, each 3min.

It is enzyme：To on nitrocellulose filter plus GAM-AP (is conjugated the sheep anti mouse secondary antibody of alkaline phosphatase, is purchased from U.S. KPL Company, similarly hereinafter), it reacts 1 hour.

Washing：With 20mM Tirs-HCl, pH8.0,0.5M NaCl, 0.05%Tween20 solution washs nitrocellulose Film 5 times, each 3min.

Colour developing：Color developing agent A, B liquid each 50ul (color developing agent A liquid is added per hole：13.4g/L Na₂HPO₄.12H₂O+4.2g/L Citric acid .H₂O+0.3g/L carbamide peroxides；Color developing agent B liquid：0.2mM/L tetramethyl benzidines (TMB)+20mM/L dimethyl methyls Amide；Similarly hereinafter), develop the color 10min.

Antibody 4B3 and the reactive WB testing results of the other HPV L1 albumen of different shaped are as shown in Figure 1.The results show that With 4B3 specific reaction can occur for the HPV L1 albumen for the 11 kinds of types tested.In addition, WB testing results are also shown, resist Body 13A10,4H4 and 12B9 cannot react with the L1 albumen of HPV6,11,16,18,31,33,45,52,58.Above-mentioned knot Fruit shows that the epitope that antibody 4B3 is identified is linear epitope, and the table that remaining 3 strain antibody (13A10,4H4,12B9) is identified Position is comformational epitope.

Positioning, identification and the homology analysis for the epitope that embodiment 2.4B3 is identified

Since the antibody 4B3 epitopes identified are linear epitope, the method for cDNA clones may be used to its epitope It is positioned.

First, by HPV16L1 albumen, cDNA clones are expressed in Escherichia coli, prepare 10 subclone peptides, each peptide altogether 65aa, and (aa indicates amino acid, and the n-th bit amino is indicated when being placed in before digital n for overlapping of the 2 adjacent peptides with 15aa Acid (for example, aa130 indicate the 130th amino acids), then indicated when being placed in after number polypeptide length be n amino acid (under Together)).This 10 subclone peptides are respectively designated as：HPV16L1A-HPV16L1J, wherein HPV16L1A corresponds to HPV16L1 The aa 1-65, the aa 51-115, HPV16L1C that HPV16L1B corresponds to HPV16L1 albumen of albumen correspond to HPV16L1 albumen Aa 101-165, and so on.

WB reactivity of this 10 subclone peptides with monoclonal antibody 4B3 is detected, the results are shown in Figure 2.The results show that HPV16L1F (aa251-aa315) it can react with monoclonal antibody 4B3 with HPV16L1G (aa301-aa365), and other subclone peptides cannot It reacts with monoclonal antibody 4B3.This shows that the epitope that monoclonal antibody 4B3 is identified is located at the position of HPV16L1F and HPV16L1FG overlappings Place, that is, be located at the aa301-315, sequence such as SEQ ID NO of HPV16L1 albumen:Shown in 41.

Further, for this 15 amino acid (SEQ ID NO:41) design has synthesized 17 segment polypeptides, is respectively designated as L1FG1-L1FG17, amino acid sequence are respectively SEQ ID NO:41-57 (synthesis of Shanghai Sheng Gong biotech firms).Then, lead to Cross the combination situation that competitive ELISA tests and analyzes these polypeptides (L1FG1-L1FG17) and monoclonal antibody 4B3.It is as follows：

The combination of polypeptide and monoclonal antibody：17 segment polypeptides are mixed with monoclonal antibody respectively, and polypeptide is excessive.Meanwhile it is right that the positive is arranged According to：15 peptides (SEQ ID NO:41)；And negative control：Only monoclonal antibody sample, and it is added without polypeptide.All samples are all in 37 DEG C It incubates 1 hour.

Coating：It will be in HPV16L1 protein adsorptions to 8K-96 orifice plates.

Sample-adding：19 samples after incubation are added to respectively and are coated in the hole of HPV16L1.

It is incubated：96 orifice plates are sealed with sealing plate film, react 30min in 37 DEG C of biochemical cultivation cases.

Washing：It carefully takes sealing plate film off, is washed 5 times with board-washing machine washing, last time button as possible is dry.

It is enzyme：100 μ l GAM-AP are added per hole.

It is incubated：96 orifice plates are sealed with sealing plate film, react 30min in 37 DEG C of biochemical cultivation cases.

Washing：It carefully takes sealing plate film off, is washed 5 times with board-washing machine washing, last time button as possible is dry.

Colour developing：Color developing agent A liquid and B liquid (seeing above) each 50 μ l, 37 DEG C of colour developing 10min are added per hole.

Result judgement：The result of sample to be tested and the result of positive control and negative control are compared.

The results show that polypeptide L1FG1-3 and L1FG10-16 can react with monoclonal antibody 4B3.These polypeptides of comprehensive analysis Amino acid sequence, the epitope that 4B3 is identified is determined as 6 peptides, sequence is SEQ ID NO:58, correspond to HPV16L1 eggs (SEQ ID NO in vain:59) aa303-308.

The homology analysis for the epitope that 4B3 is identified on HPV16L1 albumen

All HPV that will be included in the amino acid sequence and ncbi database of HPV16L1 albumen using MEGA5.0 softwares The sequence of L1 albumen is compared, and analysis corresponds to HPV16L1 albumen (SEQ ID NO:59) sequence of aa303-308 it is same Source property.Through searching for and screening, sequences of the HPV in the section of 121 types is obtained from ncbi database.To these sequences into Row compares, and for statistical analysis to the conservative of each amino acid.

Shown in analysis result Table A.The results show that the aa303-308 of HPV16L1 albumen ten code insurances in 121 HPV types Keep, wherein the 303rd, 304,305, the homologys of 307 amino acids 85% or more (the 304th, 305,307 amino acids it is same 95%) source property has been even more than；306th, the difference of 308 amino acids is relatively large, but homology still reaches 50% or more.This Illustrate, the epitope that 4B3 is identified is conserved epitope, is extremely conservative section in the HPV of multiple types, and therefore constitute The wide spectrum identification of monoclonal antibody 4B3 with combine active basis.

Table A：The sequence homology analysis for the epitope that antibody 4B3 is identified

The WB of HPV L1 albumen in embodiment 3.HPV the infected's cervical exfoliated cell is detected

Since monoclonal antibody 4B3 can react in western blot analysis with the HPV L1 of a variety of types, can incite somebody to action It is used to detect the expression of L1 albumen in patient's cervical exfoliated cell.For this purpose, tested first by western blot analysis, Determine the suitable concentration of the monoclonal antibody 4B3 for detection method；Then the detection pole of the HPV L1 of test 4B3 pairs of 11 kinds of types of monoclonal antibody Limit；Finally, by western blot analysis, 63 precancerous lesions of uterine cervixs of HPV DNA test positive are had detected with monoclonal antibody 4B3 The sample of patient.

The WB of the combination of the HPV L1 of the 4B3 of various concentration and each type is analyzed

SDS- polyacrylamide gel electrophoresises：The L1 albumen of HPV6,11,16,18,31,33,45,52,58 is diluted to 5 μ G/ml carries out SDS- polyacrylamide gel electrophoresises.

Transferring film：It will be on the albumen electrotransfer to nitrocellulose filter on SDS- polyacrylamide gels.

Closing：Nitrocellulose filter is immersed in 5% defatted milk, 60min is closed.

Add antibody:Antibody 4B3 is diluted to 10 μ g/ml, 1 μ g/ml, 0.1 μ g/ml and 0.01 μ g/ml tetra- with 5% defatted milk A concentration, then respectively from different nitrocellulose film reaction 1 hour.

Washing：With 20mM Tirs-HCl, pH8.0,0.5M NaCl, 0.05%Tween20 solution washs nitrocellulose Film 5 times, each 3min.

It is enzyme：Add GAM-AP on nitrocellulose filter, reacts 1 hour.

Washing：With 20mM Tirs-HCl, pH8.0,0.5M NaCl, 0.05%Tween20 solution washs nitrocellulose Film 5 times, each 3min.

Colour developing：Color developing agent A liquid and B liquid (seeing above) colour developing 10min are added per hole.

The results are shown in Figure 3.The results show that when the concentration of 4B3 is higher than 0.1 μ g/ml, to various other HPV L1 eggs Bai Junneng reacts.Therefore, the monoclonal antibody 4B3 of such as 1 μ g/ml can be used to carry out WB detections.

WB detectable limits of the 4B3 to the HPV L1 albumen of each type

SDS- polyacrylamide gel electrophoresises：The L1 albumen of HPV6,11,16,18,31,33,45,52,58 is diluted respectively For 7 concentration：100,33,11,3.6,1.2,0.4,0.1 μ g/ml carry out SDS- polyacrylamide gel electrophoresises.

Transferring film：It will be on the albumen electrotransfer to nitrocellulose filter on SDS- polyacrylamide gels.

Closing：Nitrocellulose filter is immersed in 5% defatted milk, 60min is closed.

Add antibody:Antibody 4B3 is diluted to 1 μ g/ml with 5% defatted milk, then with nitrocellulose film reaction 1 hour.

Washing：With 20mM Tirs-HCl, pH8.0,0.5M NaCl, 0.05%Tween20 solution washs nitrocellulose Film 5 times, each 3min.

It is enzyme：Add GAM-AP on nitrocellulose filter, reacts 1 hour.

Washing：With 20mM Tirs-HCl, pH8.0,0.5M NaCl, 0.05%Tween20 solution washs nitrocellulose Film 5 times, each 3min.

Colour developing：Color developing agent A liquid and B liquid (seeing above) colour developing 10min are added per hole.

As a result as shown in Fig. 4 and table 3.The result shows that when carrying out WB detections using 4B3, the inspection of the other L1 albumen of different shaped It surveys the limit to have differences, and between 0.4 μ g/ml-3.6 μ g/ml.

Table 3：The detectable limit of the L1 albumen of HPV6,11,16,18,31,33,45,52,58

The horizontal detection of HPV L1 albumen in cervical exfoliated cell

Sample collection：65 from Zhongshan Hospital Xiamen University's OPD of Obs ＆ Gyn (in June, 2010 in September, 2011) The DNA detections of example patient, their cervical exfoliated cell are the high-risk HPV positive.Cervical exfoliated cell is acquired respectively, is preserved In PBS solution.

Sample treatment：100 μ L of cervical exfoliated cell sample are taken, 20 μ 6 × Loading of L Buffer (12% (w/v) are added SDS, 0.6% (w/v) bromophenol blue, 0.3M Tris-HCl pH 6.8,60% (v/v) glycerine, 5% (v/v) beta -mercaptoethanol, Similarly hereinafter), mixing and in 80 DEG C of water-bath 10min.

SDS- polyacrylamide gel electrophoresises：Processed sample is subjected to SDS- polyacrylamide gel electrophoresises.

Transferring film：It will be on the albumen electrotransfer to nitrocellulose filter on SDS- polyacrylamide gels.

Closing：Nitrocellulose filter is immersed in 5% defatted milk, 60min is closed.

Add antibody:Antibody 4B3 is diluted to 0.1 μ g/ml with 5% defatted milk, it is then small with nitrocellulose film reaction 1 When.

Washing：With 20mM Tirs-HCl, pH8.0,0.5M NaCl, 0.05%Tween20 solution washs nitrocellulose Film 5 times, each 3min.

It is enzyme：Add GAM-AP on nitrocellulose filter, reacts 1 hour.

Washing：With 20mM Tirs-HCl, pH8.0,0.5M NaCl, 0.05%Tween20 solution washs nitrocellulose Film 5 times, each 3min.

Colour developing：Color developing agent A liquid and B liquid (seeing above) colour developing 10min are added per hole.

Fig. 5, which is shown, uses monoclonal antibody 4B3, by immunoblotting, detects the result of cervical exfoliated cell sample.In addition, Table 4 shows the relationship of recall rate and patient's pathology that the HPV L1 in patient samples are detected using 4B3.It can from these results To find out, monoclonal antibody 4B3 can be used for detecting the expression of the HPV L1 albumen in uterine neck patient's pathological tissue.Particularly, due to Monoclonal antibody 4B3 in combination with a variety of types HPV L1 albumen (that is, wide spectrum is reactive), therefore, to the HPV L1 albumen in sample Recall rate it is higher, be significantly better than known antibody.

Table 4：The relationship of the recall rate and patient's pathology of HPV L1 albumen

Note：CIN refers to Cervical intraepitheliaI neoplasia；Wherein, CIN1-3 is respectively represented slightly, and moderate and serious tumor become (CIN points Grade is carried out according to U.S.'s gynecatoptron and uterine neck pathology meeting (ASCCP) grade scale formulated in 2003).

Identification of 4. monoclonal antibody of embodiment to the neutralization activity of pseudovirus

This experiment passes through in pseudovirus-cell and model, detects cleaning antibody pseudovirus or significantly reduces the poison of pseudovirus The ability of power.

With in pseudovirus-cell and model identification embodiment 1 in obtain three plants of monoclonal antibodies (13A10,12B9 and 4H4) it is directed to HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52, HPV58, HPV59 cape horn fever Poison neutralization activity (about in pseudovirus-cell and model, with reference to Lu Wuxun etc., bioengineering journal, 2006, volume 22： 990-995 pages).Below for detecting 13A10 antibody to the neutralization activity of HPV16 pseudovirus, exemplary description the method.

First, by 2 times of concentration gradient doubling dilutions (maximum concentration 1mg/ml) of 13A10 antibody, then it is directed to each dilution Degree, takes 50 μ l, it is mixed with the HPV16 pseudovirus (MOI=0.1) of the suitable concentration of 50 μ l in 96 orifice plates, and 4 respectively DEG C be incubated a hour.Using the mixed liquor of pseudovirus and PBS as negative control.Then each mixed liquor is separately added into advance paving Have in 96 porocyte plates of 293FT cells (per hole about 1.5 × 10⁴293FT cells), and in carbon dioxide incubator, 37 It is cultivated 72 hours at DEG C.Later, the fluorescence intensity in each hole is detected using fluorescence plate reader (Beckman companies of the U.S.).

Using the maximum monoclonal antibody dilution factor of fluorescence intensity ratio negative control reduction at least 50% as the monoclonal antibody to type HPV's Dilution factor.If the dilution factor of monoclonal antibody be less than 20, then think its for the type HPV without neutralization activity.Above-mentioned three plants Monoclonal antibody is directed to HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52, HPV58, HPV59 cape horn fever The neutralization activity of poison is summarized in table 5 that (result is with log₁₀Dilution factor indicates).Wherein, monoclonal antibody 13A10 to HPV16, HPV31, Tetra- types of HPV33, HPV58 have cross-neutralization activity；12B9 lives to two types of HPV33, HPV58 with cross-neutralization Property；4H4 has cross-neutralization activity to tri- types of HPV16, HPV31, HPV33.These results indicate that prepared by the present invention This 3 plants of monoclonal antibodies not only have HPV L1 albumen and VLP the reactivity of wide spectrum, but also with the HPV's at least two type Cross-neutralization activity.

Table 5：The neutralization activity of the HPV pseudovirus of 11 types of each monoclonal antibody pair

The affinity analysis of 5. monoclonal antibody of embodiment

This experiment uses BIACORE3000 biosensors (GE companies of the U.S.), analyzes antigen-antibody and combines and dissociate Dynamics, calculate the binding constant k of monoclonal antibody 13A10 and various HPV VLP_a, dissociation constant k_dWith affinity K_D, To reflect the power of the antibody and the combination degree of various HPV VLP.

First, with the Acetic acid-sodium acetate buffer solution of pH 5.5 by sheep anti mouse mostly it is anti-(GAM) (be purchased from KPL companies of the U.S., under It is diluted to 40 μ g/ml together).According to the specification (Biacore3000 biosensors, GE companies of the U.S.) of manufacturer, by GAM idols It is coupled on chip, and it is 15000RU that coupling level, which is arranged,.Report that coupling is horizontal according to the result of its automatic running.The results show that Final coupling level is 15834.5RU.

Antibody 13A10 to 2 μ g/ml is diluted with HBS-EP buffer solutions.Meanwhile with HBS-EP buffer solutions by HPV6, HPV11, The VLP of HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52, HPV58, HPV59 are diluted to following each respectively Different concentration, that is, 0,1,2,4,8,16 and 32nmol/L.When detection, first by antibody sample introduction 90s, then in conjunction with 120s, dissociation 420s finally uses the glycine-HCI buffer solution regeneration chip of the pH2.2 of 20mM.It is anti-that antigen is carried out according to the specification for making quotient Dynamics/affinity analysis that body combines, also, data are analyzed with Biacore3000Evaluation softwares.Antibody 13A10 and the testing result of the affinity of the HPV VLP of each type are as shown in table 6.The results show that antibody 13A10 with The affinity of HPV16,58,31,33,35 is higher, 10^-8-10^-10Between.

Table 6：Dynamic analyses of the antibody 13A10 to the HPV VLP of multiple types

Blocking experiment of 6. monoclonal antibody of embodiment to serum-antigen binding

Blocking experiment includes following two aspects.It is on one side, first by the antibody of various concentration and antigen binding, then The degree that the combination of detection serum and antigen is blocked, that is, blocking rate of the antibody to a certain serum.It is generally believed that blocking rate is high In 50%, blocked to be high；Blocking rate is disconnected for low-resistance between 20%-50%；Blocking rate is less than 20%, not hinder It is disconnected.On the other hand it is that the investigation of above-mentioned blocking rate is carried out in the more parts of serum that same antigen generates.If blocking rate is higher than 50% Serum occupy the majority, it may be considered that the antibody be such immune serum advantage antibody, the epitope identified be for producing Dominant Epitopes (Zhaohui Wang, the A monoclonal antibody against of the antigen of such raw immune serum intact human papillomavirus type 16capsids blocks the serological reactivity of most human sera；Journal of General Virology(1997),78,2209–2215).

We have used the more parts of rabbit anteserums with the immune acquisitions of HPV VLP respectively, HPV16/18 bivalent vaccine inoculators' Serum and the serum of natural HPV infection person carry out blocking experiment, to investigate antibody in corresponding HPV VLP, HPV16/18 vaccines Whether the epitope identified in antigen and natural HPV is Dominant Epitopes, and specific experiment flow is as follows.

The preparation of ELISA Plate：

The VLP for being coated with HPV16,31,33,58 respectively on 8K-96 orifice plates (8 rows × 12 arrange) is (slow using 20mM phosphate Fliud flushing (PB, pH7.4) and 0.3M NaCl solutions, are coated with overnight at 37 DEG C).Then, using PBST (10mM PBS+0.05% Tween 20) washing plate it is primary.After plate button is done, confining liquid (casein+sucrose) is added, and 2hr are closed in 37 DEG C.Again by plate Secondary button dry doubling vacuum is drained, and ELISA Plate (holes 8x12) is obtained.

The preparation of other reagents needed for blocking experiment：

Enzyme marking reagent：With more anti-(GAM) (being purchased from KPL companies of the U.S.) of HRP label sheep anti mouses Fc, enzyme marking reagent is obtained；

Negative control：It is with the monoclonal antibody 8G12 (this laboratory voluntarily prepares preservation) of anti-Hepatitis E virus (HEV) ORF2 Negative control；

Color developing agent A liquid：13.4g/L Na₂HPO₄.12H₂O+4.2g/L citric acid .H2O+0.3g/L carbamide peroxides；

Color developing agent B liquid：3,3 ', 5,5 '-tetramethyl benzidines of 0.2mM/L (TMB)+20mM/L dimethylformamides；

Terminate liquid：2M sulfuric acid；

Concentrated cleaning solution：20x PBST.

Detection process：

With liquid：50ml concentrated cleaning solutions (20 ×) are diluted to 1000ml with distilled water or deionized water, it is spare.

Number：Sample is corresponded to microwell plate sequentially to number, and in each blocking experiment, for each type setting one Rank the negative control hole of (12).

The processing of monoclonal antibody to be measured and sample-adding:

Monoclonal antibody to be measured is diluted to 100 μ g/ml, the first hole of often row of ELISA Plate is added, then by the concentration dilution of antibody 3 The second hole is added again, and doubling dilution (that is, 3 times of a concentration of latter hole in previous hole), altogether acquisition 11 dilute backward successively Degree (3 times of serial dilutions, maximum concentration are 100 μ g/ml).Last hole addition of often going is free of the dilution of antibody.

It is incubated：With sealed membrane sealing plate, biochemical cultivation case, 37 DEG C of incubation 60min are placed it in.

Washing：It carefully takes sealing plate film off, is washed 5 times with board-washing machine washing, last time button as possible is dry.

The pretreatment of the serum or antibody that are blocked and sample-adding：

Sample is serum sample：First, the serum sample by ELISA detections for blocking experiment and corresponding type The combination situation of VLP.Then, dilute serum sample according to testing result, makes it be combined with the ELISA of the VLP of corresponding type OD values are 1 or so.By the diluted serum sample for carrying out following test.

It is incubated：With sealed membrane sealing plate, biochemical cultivation case, 37 DEG C of incubation 60min are placed it in.

Washing：It carefully takes sealing plate film off, is washed 5 times with board-washing machine washing, last time button as possible is dry.

It is enzyme：100 μ l of enzyme marking reagent are added in corresponding aperture respectively.

It is incubated：With sealed membrane sealing plate, biochemical cultivation case, 37 DEG C of incubation 45min are placed it in.

Washing：It carefully takes sealing plate film off, is washed 5 times with board-washing machine washing, last time button as possible is dry.

Colour developing：Color developing agent A liquid and B liquid each 50 μ l, 37 DEG C of colour developing 10min are added per hole.

It measures：Terminate liquid 1 is added to drip (50 μ l) per hole, then with microplate reader with Single wavelength 450nm (blank control wells need to be set) Or dual wavelength 450nm/630nm measures each hole OD values.

Sample is the specific antibody for resisting each type：Every plant of specific antibody is marked with HRP, it then directly will be through mark The antibody of note dilutes 1000 times, and in adding hole.

It is incubated：With sealed membrane sealing plate, biochemical cultivation case, 37 DEG C of incubation 60min are placed it in.

Washing：It carefully takes sealing plate film off, is washed 5 times with board-washing machine washing, last time button as possible is dry.

Colour developing：Color developing agent A liquid and B liquid each 50 μ l, 37 DEG C of colour developing 10min are added per hole.

It measures：Terminate liquid 1 is added to drip (50 μ l) per hole, then with microplate reader with Single wavelength 450nm (blank control wells need to be set) Or dual wavelength 450nm/630nm measures each hole OD values.

Result judgement：

A) normal range (NR) of negative control：Under normal circumstances, OD value≤0.1 of negative control hole is (if negative control hole OD values are more than 0.1, then should give up；If the OD values of all negative control holes are both greater than 0.1, experiment should be repeated；If negative control The OD values in hole are less than 0.03, then press 0.03 and calculate).

B) calculating of critical value (CUTOFF)：The mean value+0.15 of negative control hole OD values.

C) judgement of single hole blocking rate：By the OD values in every hole with not plus the OD values in hole of monoclonal antibody to be measured are compared, and will Blocking rate per hole is calculated as：

Blocking rate=(holes 1- OD values)/1 × 100%

D) calculating of antibody blocking rate：With a concentration of abscissa of monoclonal antibody used in each hole, it is with the blocking rate in the hole Ordinate maps and is fitted to parabola.Blocking rate when calculating antibody is a concentration of infinitely great, as the antibody to the part The blocking rate of serum.

Using the above method, antibody 13A10 is had detected respectively to the rabbit anteserum with the immune acquisition in HPV16,31,33 or 58 (each 2 parts), 4H4 is to the rabbit anteserum (each 2 parts) with the immune acquisition in HPV16,31 or 33, and 12B9 with HPV33 or 58 is immune to being obtained Rabbit anteserum (each 2 parts) blocking rate.Testing result is as shown in table 7- tables 9.

, the results show that the blocking rate of the rabbit anteserum of 13A10 couples of 2 parts of HPV58 of antibody is all higher than 50%, this shows antibody for these 13A10 is the advantage antibody of HPV58VLP immune serums, and the epitope identified is the Dominant Epitopes of HPV58VLP；Antibody 13A10 is below 50% to the blocking rate of the rabbit anteserum of HPV16,31,33, this show antibody 13A10 not and be HPV16,31, The advantage antibody of 33VLP immune serums.Similarly, antibody 4H4 and 12B9 nor HPV16,31,33,58VLP immune serums it is excellent Gesture antibody.

Table 7：Blockings of the 13A10 to the rabbit anteserum of HPV16,31,33,58

Table 8：Blockings of the 4H4 to HPV33,58 rabbit anteserum

Table 9：Blockings of the 12B9 to HPV33,58 rabbit anteserum

In addition, also having detected antibody 13A10 respectively to the serum of the more parts of natural infection persons of HPV16,33,58,4H4 is to more parts The blocking rate of the serum of the natural infection person of HPV16,31,33.Testing result is as shown in table 10- tables 11.

These the results show that the serum of 13A10 couples of 14 parts of HPV58 natural infection persons of antibody blocking rate all be higher than 50%, This shows that antibody 13A10 is the advantage antibody of the serum of HPV58 the infected, and the epitope identified is the advantage of natural HPV58 Epitope；Antibody 13A10 is below 50% to the blocking rate of the serum of HPV16,33 natural infection persons, this shows antibody 13A10 not Be HPV16,33 natural infection persons serum advantage antibody.Similarly, antibody 4H4 is nor the natural infection person of HPV16,31,33 Serum advantage antibody.

Table 10：Blockings of the 13A10 to the serum of the natural infection person of HPV16,58 or 33

Table 11：Blockings of the 4H4 to the serum of the natural infection person of HPV16,58 or 33

The separation of the light chain gene and heavy chain gene variable region of 7. monoclonal antibody of embodiment

Half adhere-wall culture 10⁷A hybridoma blows afloat adherent cell with blowpipe and is allowed to suspend, and is transferred into new 4ml centrifuge tubes in.3min is centrifuged with 1500rpm, collects the cell of precipitation, and be resuspended in the 100 sterile PBS of μ l (pH7.45) in.Cell suspension is transferred in a new 1.5ml centrifuge tubes, 800 μ l Trizol of addition (Roche, Germany), mixing and is gently overturned, 10min is stood.Then, 200 μ l chloroforms are added and acutely vibrate 15s, stand 10min. Later, 15min is centrifuged, and is shifted in supernatant liquid to a new 1.5ml centrifuge tubes with 4 DEG C, 12000rpm, be added isometric Isopropanol, mixing simultaneously stand 10min.Later, 10min is centrifuged with 4 DEG C, 12000rpm, abandons supernatant；600 μ l, 75% ethyl alcohol is added It is washed, 5min is centrifuged with 4 DEG C, 12000rpm, abandons supernatant；60 DEG C of vacuum will be deposited in and drain 5min.It later, will be transparent Precipitation is dissolved in 70 μ l DEPC H₂In O, and it is distributed into two pipes.Often pipe is separately added into 1 μ l reverse transcription primers, wherein what a pipe was added Reverse transcription primer is MVJkR (5'-CCg TTT (T/g) AT (T/C) TC CAg CTT ggT (g/C) CC-3'), light for expanding Chain variable region gene；The reverse transcription primer that another pipe is added is MVDJhR (5'-C ggT gAC Cg (T/A) ggT (C/g/T) CC TTg (g/A) CC CCA-3'), for expanding heavy chain variable region gene.1 μ l dNTP (Shanghai life work) are added into every pipe, then In 72 DEG C of water-bath 10min, it is immediately placed on 5min in ice bath later；Then 10 μ l 5x reverse transcription buffers, 1 μ l AMV is added (10u/ μ l, Pormega), 1 μ l Rnasin (40u/ μ l, Promega), after mixing in 42 DEG C by RNA reverse transcriptions at cDNA.

Using PCR (PCR) method separation antibody gene variable region, the Ig- according to Novagen companies is used The primer sets (table 12) of Prime kits synthesis and two other downstream primer MVJkR and MVDJhR (Shanghai Bo Ya companies Synthesis), wherein MVJkR is the downstream primer expanded for chain variable region gene, and MVDJhR is for heavy chain variable region gene The downstream primer of amplification.Used template is the cDNA obtained by the above method.PCR conditions are：94℃5min；50 are followed (the 94 DEG C of 40s, 53 DEG C of 1min, 72 DEG C of 50s) of ring；72℃15min.Target fragment is recycled, and is cloned into pMD 18-T carriers, Then it is sequenced (Shanghai Bo Ya companies).Blast comparisons are carried out to sequencing sequence, to determine the nucleotides sequence of antibody variable region Row, and corresponding amino acid sequence is determined in turn.

According to the method described above, it is cloned from hybridoma cell strain 4B3,13A10,12B9,4H4 secreted by each cell strain The variable region gene of monoclonal antibody, and corresponding amino acid sequence is determined.Table 12 shows the sequence of used sense primer.Table 13 show the heavy chain of 4 plants of monoclonal antibodies and the nucleotide sequence of light chain variable region and the sequence number of amino acid sequence.Table 14 show according to Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)) described in 4 plants of monoclonals determining of method it is anti- The cdr amino acid sequence of body.

Table 12：Sequence for the sense primer for expanding monoclonal antibody variable region gene

Table 13：The sequence number of the variable region of 4 plants of monoclonal antibodies

Table 14：The cdr amino acid sequence of 4 plants of monoclonal antibodies

Various genetic engineering antibodies, example can be prepared by known antibody engineering technology using the sequence of above-mentioned identification Such as chimeric antibody, humanized antibody, single-chain antibody, double antibody etc., and retain the biological characteristics for the monoclonal antibody that it is originated from Property (for example, the wide spectrum to HPV L1 albumen is reactive, and/or active to the cross-neutralization of the HPV of at least two type).

Although the specific implementation mode of the present invention has obtained detailed description, it will be appreciated by those skilled in the art that：Root According to all introductions having disclosed, details can be carry out various modifications and be changed, and these change the guarantor in the present invention Within the scope of shield.The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims (39)

1. the L1 albumen of the HPV of multiple types and/or the monoclonal antibody of VLP or its antigen binding fragment can be specifically bound Section, wherein the monoclonal antibody includes：

Including amino acid sequence is SEQ ID NO:The VH of the CDR of 35-37, and comprising amino acid sequence be SEQ ID NO:38- The VL of 40 CDR；

The antigen-binding fragment is selected from Fab, Fab', F (ab')₂, Fv, single-chain antibody and double antibody.

2. the monoclonal antibody of claim 1 or its antigen-binding fragment, wherein the monoclonal antibody includes：

Such as SEQ ID NO:VH shown in 14 and such as SEQ ID NO:VL shown in 16.

3. the monoclonal antibody of any one of claim 1-2 or its antigen-binding fragment, wherein the monoclonal antibody is selected from people Source antibody and chimeric antibody.

4. the monoclonal antibody of claim 1 or its antigen-binding fragment, wherein the single-chain antibody is scFv.

5. the monoclonal antibody of any one of claim 1-2 or its antigen-binding fragment, wherein the monoclonal antibody is with small In 10^-5The K of M_DIn conjunction with the L1 albumen or VLP of HPV.

6. the monoclonal antibody of any one of claim 1-2 or its antigen-binding fragment, wherein the monoclonal antibody can The L1 albumen and VLP of HPV of the specific binding selected from following one or more types：HPV16、HPV31、HPV33、HPV35 And HPV52.

7. the monoclonal antibody of any one of claim 1-2 or its antigen-binding fragment, wherein during the monoclonal antibody is And antibody, the HPV selected from following one or more types can be neutralized：HPV16, HPV31 and HPV33.

8. the monoclonal antibody of any one of claim 1-2 or its antigen-binding fragment, wherein the monoclonal antibody includes Non- CDR region, and the non-CDR region is from the species for not being muroid.

9. the monoclonal antibody of claim 8 or its antigen-binding fragment, wherein the non-CDR region comes from human antibody.

10. the monoclonal antibody of any one of claim 1-2 or its antigen-binding fragment, wherein the monoclonal antibody is The monoclonal antibody generated by hybridoma cell strain 4H4, the hybridoma cell strain 4H4 are preserved in China typical culture collection Center (CCTCC), and there is preserving number CCTCC-C201265.

11. separation nucleic acid molecules, it includes be capable of encoding antibody heavy variable region nucleic acid sequence and can encoding antibody it is light The nucleic acid sequence of chain variable region, wherein the heavy chain of antibody variable region includes：Amino acid sequence is SEQ ID NO:35-37's CDR；The antibody light chain variable region includes：Amino acid sequence is SEQ ID NO:The CDR of 38-40.

12. the nucleic acid molecules of the separation of claim 11, wherein the heavy chain of antibody variable region has SEQ ID NO:Shown in 14 Amino acid sequence.

13. the nucleic acid molecules of the separation of claim 11, wherein the nucleic acid sequence tool for capableing of encoding antibody heavy variable region There are SEQ ID NO:Nucleotide sequence shown in 13.

14. the nucleic acid molecules of the separation of claim 11, wherein the antibody light chain variable region has SEQ ID NO:Shown in 16 Amino acid sequence.

15. the nucleic acid molecules of the separation of claim 11, wherein the nucleic acid sequence tool for capableing of encoding antibody light variable region There are SEQ ID NO:Nucleotide sequence shown in 15.

16. a kind of carrier, it includes the nucleic acid molecules of the separation of any one of claim 11-15.

17. a kind of host cell, it includes the nucleic acid molecules of the separation of any one of claim 11-15 or claims 16 Carrier.

18. preparing the monoclonal antibody of any one of claim 1-10 or the method for its antigen-binding fragment comprising, suitable Under conditions of cultivate the host cell of claim 17, and the monoclonal antibody or its antigen knot are recycled from cell culture Close segment.

19. hybridoma cell strain 4H4 is preserved in China typical culture collection center (CCTCC), and has preserving number CCTCC-C201265.

20. kit comprising the monoclonal antibody of any one of claim 1-10 or its antigen-binding fragment.

21. the kit of claim 20, wherein the monoclonal antibody or its antigen-binding fragment further include detectable mark Note.

22. the kit of claim 21, wherein the detectable label is selected from radioactive isotope, and luminescent substance is coloured Substance and enzyme.

23. the kit of claim 21, wherein it is described it is detectable label be.

24. the kit of claim 20, wherein the kit further includes secondary antibody, Dan Ke described in specific recognition Grand antibody or its antigen-binding fragment；Optionally, the secondary antibody further includes detectable label.

25. the kit of claim 24, wherein the detectable label is selected from radioactive isotope, and luminescent substance is coloured Substance and enzyme.

26. the kit of claim 24, wherein it is described it is detectable label be.

27. the method for detecting HPV L1 albumen or VLP presence in the sample or its horizontal non-diagnostic purpose comprising Use the monoclonal antibody or its antigen-binding fragment of any one of claim 1-10.

28. the method for claim 27, wherein the monoclonal antibody or its antigen-binding fragment further include detectable mark Note.

29. the method for claim 28, wherein the detectable label is selected from radioactive isotope, luminescent substance, colored substance Matter and enzyme.

30. the method for claim 28, wherein it is described it is detectable label be.

31. the method for claim 27, wherein the method further includes being come using the secondary antibody for carrying detectable label Detect the monoclonal antibody or its antigen-binding fragment.

32. the method for claim 31, wherein the detectable label is selected from radioactive isotope, luminescent substance, colored substance Matter and enzyme.

33. the method for claim 31, wherein it is described it is detectable label be.

34. the purposes of the monoclonal antibody of any one of claim 1-10 or its antigen-binding fragment in reagent preparation box, institute Kit is stated for detecting HPV L1 albumen or VLP presence in the sample or it is horizontal, or for diagnosing whether subject feels HPV is contaminated.

35. a kind of pharmaceutical composition, it includes the monoclonal antibody of any one of claim 1-10 or its antigen-binding fragment, with And pharmaceutically acceptable carrier and/or excipient.

36. the method for neutralizing the non-treatment purpose of the virulence of HPV in sample comprising, by sample and right comprising HPV It is required that the monoclonal antibody of any one of 1-10 or the contact of its antigen-binding fragment.

37. the monoclonal antibody of any one of claim 1-10 or its antigen-binding fragment are used to prepare the purposes of drug, described Drug is used to neutralize the virulence of HPV in sample.

38. the use of the monoclonal antibody of any one of claim 1-10 or its antigen-binding fragment in preparing pharmaceutical composition On the way, described pharmaceutical composition be used for prevent or treat subject HPV infection or with the relevant disease of HPV infection.

39. the purposes of claim 38, wherein the described and relevant disease of HPV infection is cervical carcinoma.