CN105175537B - The broad-spectrum monoclonal antibody of anti-HPV L1 albumen or its antigen-binding fragment and their purposes - Google Patents

The broad-spectrum monoclonal antibody of anti-HPV L1 albumen or its antigen-binding fragment and their purposes Download PDF

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CN105175537B
CN105175537B CN201510710642.5A CN201510710642A CN105175537B CN 105175537 B CN105175537 B CN 105175537B CN 201510710642 A CN201510710642 A CN 201510710642A CN 105175537 B CN105175537 B CN 105175537B
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antibody
monoclonal antibody
seq
hpv
antigen
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CN105175537A (en
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李少伟
肖洁琼
魏旻希
陆剑
俞海
张军
夏宁邵
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Xiamen University
Xiamen Innovax Biotech Co Ltd
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Xiamen University
Xiamen Innovax Biotech Co Ltd
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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 that are diagnosed, prevented or 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 technique
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 50-55nm's by the diameter that Major capsid protein L1 and secondary capsid protein L2 are formed Regular dodecahedron particle.The HPV of more than 100 kinds of types is had now been found that, wherein can be with tumorigenic relationship according to it Be 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 prevention 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 vaccine researched and developed is mostly with virus-like particle for main vaccine composition.However, the study found that HPV VLP mainly induces the neutralizing antibody for homotype HPV, generates the protective immunity for being directed to homotype HPV, and only some same There are cross protection (Giroglou T, Sapp M, Fl igge 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 research shows that the antibody that HPV L1 albumen is induced It is mostly type specificity neutralizing antibody, the rarely report of the wide spectrum neutralizing antibody across type.At present, it has been found that across type Antibody only have this 3 plants of cross-neutralization monoclonal antibodies of HPV16J4, HPV16I23 and HPV33E12, 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 lower, than specificity neutralization monoclonal antibody (such as: 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 applied in the diagnosis of HPV, prevention and treatment.
Summary of the invention
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 that are diagnosed, prevented or 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 (see, e.g. NCBI GENBANK database serial number: DQ469930.1) well known in the art.In this application, when referring to When the L1 albumen or VLP of HPV, relate generally to selected from following 11 kinds of types HPV L1 albumen or VLP: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 referring 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 residue of HPV16 L1 albumen.However, it is as used herein, Term " HPV L1 albumen " be intended to include the other HPV of all models (especially above-mentioned 11 kinds of types) L1 albumen.Therefore, it states " the 303-308 amino acids residue of HPV L1 albumen " not only the 303-308 amino acids including HPV16 L1 albumen are residual Base, and the corresponding sequence segment in the HPV L1 albumen including other types.
As used herein, when referring to the amino acid sequence of HPV16L1 albumen, referring to the ammonia of SEQ ID NO:59 Base acid sequence is described.For example, statement " the 303-308 amino acids residue of HPV16 L1 albumen " refers to, SEQ ID The 303-308 amino acids residue of amino acid sequence shown in NO:59.However, it will be appreciated by those skilled in the art that in HPV16 L1 In the amino acid sequence of albumen, can it is naturally-produced or be artificially introduced mutation or variation (including but not limited to, replace, missing and/or Addition), without influencing its biological function.Therefore, in the present invention, term " HPV16 L1 albumen " should include all such sequences Column, including sequence shown in such as SEQ ID NO:59 and its natural or artificial variant.Also, when description HPV16L1 egg When white sequence fragment, not only includes the sequence fragment of SEQ ID NO:59, further include the natural or people of SEQ ID NO:59 Corresponding sequence segment in work variant.For example, statement " the 303-308 amino acids residue of 16 L1 albumen of HPV " includes, Respective segments in the 303-308 amino acids residue of SEQ ID NO:59 and its variant (natural or artificial).According to this Invention, statement " corresponding sequence segment " or " respective segments " refer to, when carrying out optimal comparison to sequence, i.e., when sequence is compared When to obtaining highest percentage identity, the segment of equivalent site is located in the sequence that is compared.
As used herein, term " antibody " refers to, refers 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 (VH) and heavy chain constant region (CH) composition.Heavy chain constant region is by 3 structures Domain (CH1、CH2 and CH3) it forms.Each light chain is by light chain variable region (VL) and constant region of light chain (CL) composition.Constant region of light chain is by one A domain CLComposition.The constant region of antibody can mediated immunity globulin and host tissue or the factor, including each of immune system The combination of the first component (C1q) of kind cell (for example, effector cell) and classical complement system.VHAnd VLArea can be also subdivided into With denatured region (referred to as complementary determining region (CDR)), it is interspersed with the more conservative region for being known as framework region (FR). Each VHAnd VLBy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 arranged from amino terminal to carboxyl terminal 3 A CDR and 4 FR composition.Variable region (the V of each heavy chain/light chain pairHAnd VL) 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. (1987 and 1991)) or Chothia&Lesk (1987)J.Mol.Biol.196:901-917；The definition of Chothia et al. (1989) Nature 342:878-883.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, the ability for the same antigen for keeping specific binding full length antibody to be combined, and/or compete with full length antibody to antigen 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 segment " means by VHAnd CHThe antibody fragment of 1 structural domain composition；Term " Fv Segment " means by the V of the single armed of antibodyLAnd VHThe antibody fragment of structural domain composition；Term " dAb segment " means by VHStructural domain The antibody fragment (Ward et al., Nature 341:544-546 (1989)) of composition；Term " Fab segment " means by VL、VH、CL And CHThe 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 VLAnd VHStructural 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 molecule can have general structure: NH2-VLConnector-VH- COOH or NH2-VHConnector-VL-COOH.It closes Suitable prior art connector is made of duplicate GGGGS amino acid sequence or its variant.For example, can be used has amino acid sequence (GGGGS)4Connector, but can also be used its variant (Holliger et al. (1993), Proc.Natl.Acad.Sci.USA 90: 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 VHAnd VLStructural 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 by with for complete antibody in a manner of identical mode with regard to specificity screening The antigen-binding fragment of antibody.
It herein, unless clearly indicated by the context, not only include complete anti-otherwise when referring to term " antibody " Body, and the antigen-binding fragment including 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. is reported for the first time and 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 such antibody, a part of 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 (4,816,567 to Cabilly et of U.S.P al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984)).
As used herein, term " humanized antibody " refers to, the whole of source of people immunoglobulin (receptor antibody) Or 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, which can be, has expected specificity, compatibility or reactive non-source of people (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 " refers 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, is combined on antigen by immunoglobulin or antibody specificity Position." epitope " is also referred to as " antigenic determinant " in the art.Epitope or antigenic determinant are usually by the chemical activity 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 protein separated from each other Amino acid residue and exist.
As used herein, term " epitope peptide " refers to, can be used as the peptide fragment of epitope on antigen.In some cases Under, individual epitope peptide can be directed to antibody specificity identification/combination of 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, so that the epitope peptide can be identified by antibody or immune system.Examples of such carriers egg It is white be it is well known to those skilled in the art, 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)L1 particles presenting the common neutralizing epitope for the L2 minor capsid protein of HPV-6 and 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 end 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.Ratti et 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.The high throughput method that the antibody in conjunction with same epitope is obtained based on their cross competition is described in the world In patent application WO 03/48731.Therefore, routine techniques well known by persons skilled in the art can be used, obtain and of the invention 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 locating for it Changed, or isolate the substance under natural surroundings, or both situation have generation.For example, a certain living animal body It is interior it is naturally occurring certain not by isolated polynucleotide or polypeptide, 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 ", which refers to, is made of Escherichia coli (bacterial strain) with carrier 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 for carrying it obtains 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, such as the artificial chromosome (PAC) of yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or the source P1；Phagocytosis Body such as λ bacteriophage or M13 bacteriophage and animal virus etc..The animal virus that can be used as carrier includes but is 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, can be used for importing the cell of carrier comprising but it is unlimited In, such as the prokaryotic cell of Escherichia coli or withered grass bacterium, such as the fungal cell of yeast cells or Aspergillus, such as S2 drosophila cell Or the insect cell of Sf9 etc., or such as fibroblast, Chinese hamster ovary celI, COS cell, NSO cell, HeLa cell, bhk cell, The zooblast of 293 cell of HEK or people's cell etc..
As used herein, term " identity " 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 that is 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% identity.For example, DNA sequence dna CTGACT and CAGGTT shares 50% identity (having 3 location matches in 6 positions in total).In general, by two sequences It is compared when comparing to generate maximum identity.Such comparison can be by using for example, can be by computer program for example Needleman that Align program (DNAstar, Inc.) easily carries 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 program (version 2 .0) also can be used Biosci., (1988) 4:11-17) algorithm, use PAM120 weight residue table (weight residue table), 12 Gap Length Penalty and 4 Gap Penalty measure the percentage identity between two amino acid sequences.In addition, can be used The Needleman and Wunsch (J MoI being integrated into the GAP program of GCG software package (can be obtained on www.gcg.com) Biol.48:444-453 (1970)) algorithm, use 62 matrix of Blossum or PAM250 matrix 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 identity.
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, including formed covalent bond or the ability of hydrogen bond etc.) residue carry out displacement.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 block (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., Brummel l 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, body can be stimulated to form spy The ability of xenoantibody or sensitized lymphocyte.It both referred to that 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, 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 (KD) combine the antigen.
As used herein, term " KD" refer to specific antibodies-antigen interactions Dissociation equilibrium constant, it uses 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 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 constant (KD) 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 that 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 invention being capable of specificity knowledges The L1 albumen and VLP of not/at least five kinds of types of combination HPV.
As used herein, term " monoclonal antibody " and " monoclonal antibody " have the same meaning and are used interchangeably； Term " polyclonal antibody " and " how anti-" have the same meaning and are 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 It 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 Carrier and/or excipient physiologically 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 is not limited to: pH adjusting agent, surface Activating agent, adjuvant, ionic strength reinforcing agent.For example, pH adjusting agent includes but is 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 assistant Agent is then using more in clinical trial.
As used herein, term " HPV VLP " refers to, is self-assembly of by the HPV L1 albumen of vivoexpression Virus-like particle.
As used herein, term " HPV pseudovirus " refers to, 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 what intracellular episome viral DNA or external source imported 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, HPV 33, HPV 35, HPV 45, HPV 52, HPV 58,59 pseudovirus of HPV.
As used herein, term " effective quantity ", which refers to, is enough to obtain or at least partly obtain desired effect Amount.For example, prevention disease (such as HPV infection or disease relevant to HPV infection) effective quantity refers to, it is sufficient to prevent, prevents, or Postpone the amount of the generation of disease (such as HPV infection or disease relevant to HPV infection)；Treatment condition effective amount refers to, it is sufficient to Cure or at least partly prevent suffer from disease patient disease and its complication amount.Such effective quantity is measured to exist completely Within the limit of power of those skilled in the art.For example, therapeutical uses, which are effectively measured, will depend on disease to be treated Severity, the overall status of the immune system of patient oneself, the ordinary circumstance of patient such as age, weight and gender, drug Method of application, and the other treatment being administered simultaneously etc..
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 selected from following heavy chain variable region (VH):
(1) VH comprising the CDR that amino acid sequence is SEQ ID NO:17-19；
(2) VH comprising the CDR that amino acid sequence is SEQ ID NO:23-25；
(3) VH comprising the CDR that amino acid sequence is SEQ ID NO:29-31；With
(4) VH comprising the CDR that amino acid sequence is SEQ ID NO:35-37, and/or
The monoclonal antibody includes selected from following light chain variable region (VL):
(1) VL comprising the CDR that amino acid sequence is SEQ ID NO:20-22；
(2) VL comprising the CDR that amino acid sequence is SEQ ID NO:26-28；
(3) VL comprising the CDR that amino acid sequence is SEQ ID NO:32-34；With
(4) VL comprising the CDR that amino acid sequence is SEQ ID NO:38-40.
In a preferred embodiment, the monoclonal antibody includes selected from following heavy chain variable region (VH):
(1) VH as shown in SEQ ID NO:2；
(2) VH as shown in SEQ ID NO:6；
(3) VH as shown in SEQ ID NO:10；With
(4) VH as shown in SEQ ID NO:14.
In a preferred embodiment, the monoclonal antibody includes selected from following light chain variable region (VL):
(1) VL as shown in SEQ ID NO:4；
(2) VL as shown in SEQ ID NO:8；
(3) VL as shown in SEQ ID NO:12；With
(4) VL as shown in SEQ ID NO:16.
In a preferred embodiment, the monoclonal antibody includes:
(1) comprising amino acid sequence be SEQ ID NO:17-19 CDR VH, and comprising amino acid sequence be SEQ ID The VL of the CDR of NO:20-22；
(2) comprising amino acid sequence be SEQ ID NO:23-25 CDR VH, and comprising amino acid sequence be SEQ ID The VL of the CDR of NO:26-28；
(3) comprising amino acid sequence be SEQ ID NO:29-31 CDR VH, and comprising amino acid sequence be SEQ ID The VL of the CDR of NO:32-34；Or
(4) comprising amino acid sequence be SEQ ID NO:35-37 CDR VH, and comprising amino acid sequence be SEQ ID The VL of the CDR of NO:38-40.
In a preferred embodiment, the monoclonal antibody includes:
(1) VH as shown in SEQ ID NO:2, and the VL as shown in SEQ ID NO:4；
(2) VH as shown in SEQ ID NO:6, and the VL as shown in SEQ ID NO:8；
(3) VH as shown in SEQ ID NO:10, and the VL as shown in SEQ ID NO:12；Or
(4) VH as shown in SEQ ID NO:14, and the VL as shown in SEQ ID NO: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 deposit 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 KDIn 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, monoclonal antibody 4B3 of the invention can specifically bind the HPV of 11 kinds of types L1 albumen and VLP；Monoclonal antibody 13A10 of the invention can specifically bind 6 kinds of types HPV (HPV16, HPV31, HPV33, HPV35, HPV52 and HPV58) L1 albumen and VLP；Monoclonal antibody 4H4 of the invention can specifically bind 5 kinds of types The L1 albumen and VLP of HPV (HPV16, HPV31, HPV33, HPV35 and HPV52)；Monoclonal antibody 12B9 of the 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: the VH comprising the CDR that amino acid sequence is SEQ ID NO:23-25, and/or be comprising amino acid sequence The VL of the CDR of SEQ ID NO:26-28.Preferably, such monoclonal antibody includes, the VH as shown in SEQ ID NO:6 and/or The VL as shown in SEQ ID NO: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: comprising amino acid sequence be SEQ ID NO:29-31 CDR VH, and/or comprising amino acid sequence be SEQ The VL of the CDR of ID NO:32-34.Preferably, such monoclonal antibody includes, the VH as shown in SEQ ID NO:10 and/or such as VL shown in SEQ ID NO: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: comprising amino acid sequence be SEQ ID NO:35-37 CDR VH, and/or comprising amino acid sequence be SEQ The VL of the CDR of ID NO:38-40.Preferably, such monoclonal antibody includes, the VH as shown in SEQ ID NO:14 and/or such as VL shown in SEQ ID NO: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 It is excellent from least the 50% of the combination of following monoclonal antibody, preferably at least 60%, preferably at least 70%, preferably at least 80% 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 deposit 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 deposit 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 deposit 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 deposit 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, so that 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:A Laboratory Manual, Cold Spring Harbor can be used 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: that elder generation is pre-coated on microwell plate antigen, 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 under the test antibodies of different dilutions after washing, 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 integrated 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.
List can be prepared using the hybridoma preparation method that Kohler etc. is reported in Nature 256:495 (1975) 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 use Freund's adjuvant or MPL- TDM etc..Animal can generate the lymphocyte of the antibody of secretion specific binding immunogene after receiving to be immunized in vivo.Collect mesh Lymphocyte, and it is merged with myeloma cell with suitable fusion agent (such as PEG), 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 for being able to suppress 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 medium) 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 medium to wait energy Power.Wherein, myeloma cell's first choice source of mouse myeloma, such as derivative strain (the THE Salk of MOP-21 and MC-11 mouse tumor 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 measures the binding specificity of the monoclonal antibody of hybridoma generation: immunoprecipitation or it is external combine test, 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 be grown in animal body 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.By resulting DNA molecular be inserted into expression vector in, then transfection host cell (such as E.col i cell, COS cell, 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 isolated nucleic acid molecules, encode monoclonal antibody or its antigen binding fragment of the invention Section.Such nucleic acid molecules can be isolated from hybridoma, also can use genetic engineering recombinant technique or chemistry closes It is obtained at method.
In one aspect, the present invention provides isolated nucleic acid molecules, and it includes be capable of encoding antibody heavy variable region Nucleic acid sequence, wherein the antibody heavy chain variable region includes:
(1) amino acid sequence is the CDR of SEQ ID NO:17-19；
(2) amino acid sequence is the CDR of SEQ ID NO:23-25；
(3) amino acid sequence is the CDR of SEQ ID NO:29-31；Or
(4) amino acid sequence is the CDR of SEQ ID NO:35-37.
In a preferred embodiment, the antibody heavy chain variable region has SEQ ID NO:2, SEQ ID NO:6, Amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:14.
In a preferred embodiment, the nucleic acid molecules have SEQ ID NO:1, SEQ ID NO:5, SEQ ID Nucleotide sequence shown in NO:9 or SEQ ID NO:13.
On the other hand, the present invention provides isolated nucleic acid molecules, and it includes being capable of encoding antibody light variable region Nucleic acid sequence, wherein the antibody's light chain variable region includes:
(1) amino acid sequence is the CDR of SEQ ID NO:20-22；
(2) amino acid sequence is the CDR of SEQ ID NO:26-28；
(3) amino acid sequence is the CDR of SEQ ID NO:32-34；With
(4) amino acid sequence is the CDR of SEQ ID NO:38-40.
In a preferred embodiment, the antibody's light chain variable region has SEQ ID NO:4, SEQ ID NO:8, Amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:16.
In a preferred embodiment, the nucleic acid molecules have SEQ ID NO:3, SEQ ID NO:7, SEQ ID Nucleotide sequence shown in NO:11 or SEQ ID NO:15.
On the other hand, the present invention provides a kind of carriers, and it includes isolated nucleic acid molecules of the invention.The present invention Carrier can be cloning vector, be also possible to expression vector.
In a preferred embodiment, carrier of the invention is such as plasmid, clay, bacteriophage, coemid etc..
On the other hand, the host cell comprising isolated nucleic acid molecules or carrier of the invention is additionally provided.It is such Host cell includes but is 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.).Cell of the invention may be used also To be cell line, such as 293T cell.
On the other hand, the method for preparing monoclonal antibody or its antigen-binding fragment of the invention is additionally provided, Including cultivating host cell of the 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 be selected from following hybridoma cell strain:
(1) hybridoma cell strain 4B3 is preserved in China typical culture collection center (CCTCC), and has deposit number CCTCC-C201264；
(2) hybridoma cell strain 4H4 is preserved in China typical culture collection center (CCTCC), and has deposit 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 of the heavy chain variable region of monoclonal antibody 4B3 such as SEQ ID NO:2 institute Show (its Exemplary nucleotide sequences is as shown in SEQ ID NO:1), and the amino acid sequence of light chain variable region such as SEQ ID NO:4 Shown (its Exemplary nucleotide sequences is as shown in SEQ ID NO:3).
The amino acid sequence of CDR1, CDR2 and CDR3 of monoclonal antibody 4B3 heavy chain 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 of the heavy chain variable region of monoclonal antibody 13A10 such as SEQ ID NO:6 Shown (its Exemplary nucleotide sequences is as shown in SEQ ID NO:5), and the amino acid sequence of light chain variable region such as SEQ ID (its Exemplary nucleotide sequences is as shown in SEQ ID NO:7) shown in NO:8.
The amino acid sequence of CDR1, CDR2 and CDR3 of monoclonal antibody 13A10 heavy chain 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 of the heavy chain variable region of monoclonal antibody 12B9 such as SEQ ID NO:10 Shown (its Exemplary nucleotide sequences is as shown in SEQ ID NO:9), and the amino acid sequence of light chain variable region such as SEQ ID (its Exemplary nucleotide sequences is as shown in SEQ ID NO:11) shown in NO:12.
The amino acid sequence of CDR1, CDR2 and CDR3 of monoclonal antibody 12B9 heavy chain 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 of the heavy chain variable region of monoclonal antibody 4H4 such as SEQ ID NO:14 Shown (its Exemplary nucleotide sequences is as shown in SEQ ID NO:13), and the amino acid sequence of light chain variable region such as SEQ ID (its Exemplary nucleotide sequences is as shown in SEQ ID NO:15) shown in NO:16.
The amino acid sequence of CDR1, CDR2 and CDR3 of monoclonal antibody 4H4 heavy chain 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, and specific recognition is of the invention 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 monoclonal antibody or its antigen-binding fragment of the 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 detecting monoclonal antibody or its antigen knot of the invention 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 also includes being detected using the secondary antibody for carrying detectable label Monoclonal antibody of the invention or its antigen-binding fragment.
On the other hand, monoclonal antibody or its antigen-binding fragment of the invention are provided in reagent preparation box Purposes, the presence or its level that the kit is used to detect HPV L1 albumen or VLP in the sample, or for diagnosing subject Whether HPV has been infected.
In a preferred embodiment, the monoclonal antibody is such antibody comprising: it include amino acid sequence It is classified as the VH of the CDR of SEQ ID NO:17-19, and/or the VL comprising the CDR that amino acid sequence is SEQ ID NO:20-22；It is excellent Selection of land comprising: the VH as shown in the SEQ ID NO:2 and/or VL as shown in SEQ ID NO: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: it include amino acid sequence It is classified as the VH of the CDR of SEQ ID NO:23-25, and/or the VL comprising the CDR that amino acid sequence is SEQ ID NO:26-28；It is excellent Selection of land comprising: the VH as shown in the SEQ ID NO:6 and/or VL as shown in SEQ ID NO: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: it include amino acid sequence It is classified as the VH of the CDR of SEQ ID NO:29-31, and/or the VL comprising the CDR that amino acid sequence is SEQ ID NO:32-34；It is excellent Selection of land comprising: the VH as shown in the SEQ ID NO:10 and/or VL as shown in SEQ ID NO: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: it include amino acid sequence It is classified as the VH of the CDR of SEQ ID NO:35-37, and/or the VL comprising the CDR that amino acid sequence is SEQ ID NO:38-40；It is excellent Selection of land comprising: the VH as shown in the SEQ ID NO:14 and/or VL as shown in SEQ ID NO: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 monoclonal antibody of the 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: the VH comprising the CDR that amino acid sequence is SEQ ID NO:23-25, and/or packet VL containing the CDR that amino acid sequence is SEQ ID NO:26-28；Preferably comprising: the VH as shown in SEQ ID NO:6 and/ Or the VL as shown in SEQ ID NO:8；It is highly preferred that it is monoclonal antibody 13A10；
(2) monoclonal antibody comprising: the VH comprising the CDR that amino acid sequence is SEQ ID NO:29-31, and/or packet VL containing the CDR that amino acid sequence is SEQ ID NO:32-34；Preferably comprising: the VH as shown in SEQ ID NO:10 And/or the VL as shown in SEQ ID NO:12；It is highly preferred that it is monoclonal antibody 12B9；Or
(3) monoclonal antibody comprising: the VH comprising the CDR that amino acid sequence is SEQ ID NO:35-37, and/or packet VL containing the CDR that amino acid sequence is SEQ ID NO:38-40；Preferably comprising: the VH as shown in SEQ ID NO:14 And/or the VL as shown in SEQ ID NO: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 with this need apply prevention or therapeutically effective amount this hair Bright pharmaceutical composition.
On the other hand, monoclonal antibody or its antigen-binding fragment of the invention are provided in preparation pharmaceutical composition In purposes, described pharmaceutical composition be used for prevent or treat subject HPV infection or disease (example relevant to HPV infection Such as cervical carcinoma).
In a preferred embodiment, the monoclonal antibody is such antibody comprising: it include amino acid sequence It is classified as the VH of the CDR of SEQ ID NO:23-25, and/or the VL comprising the CDR that amino acid sequence is SEQ ID NO:26-28；It is excellent Selection of land comprising: the VH as shown in the SEQ ID NO:6 and/or VL as shown in SEQ ID NO: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: it include amino acid sequence It is classified as the VH of the CDR of SEQ ID NO:29-31, and/or the VL comprising the CDR that amino acid sequence is SEQ ID NO:32-34；It is excellent Selection of land comprising: the VH as shown in the SEQ ID NO:10 and/or VL as shown in SEQ ID NO: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: it include amino acid sequence It is classified as the VH of the CDR of SEQ ID NO:35-37, and/or the VL comprising the CDR that amino acid sequence is SEQ ID NO:38-40；It is excellent Selection of land comprising: the VH as shown in the SEQ ID NO:14 and/or VL as shown in SEQ ID NO: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 monoclonal antibody of the 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 mentions Monoclonal antibody or its antigen-binding fragment of the invention 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: the VH comprising the CDR that amino acid sequence is SEQ ID NO:23-25, and/or include amino acid sequence It is classified as the VL of the CDR of SEQ ID NO:26-28；Preferably comprising: the VH as shown in SEQ ID NO:6 and/or such as SEQ ID VL shown in NO: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: comprising amino acid sequence be SEQ ID NO:29-31 CDR VH, and/or comprising amino acid sequence be SEQ The VL of the CDR of ID NO:32-34；Preferably comprising: the VH as shown in SEQ ID NO: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: the VH comprising the CDR that amino acid sequence is SEQ ID NO:35-37, and/or comprising amino acid sequence be The VL of the CDR of SEQ ID NO:38-40；Preferably comprising: the VH as shown in SEQ ID NO:14 and/or such as SEQ ID VL shown in NO:16；It is highly preferred that it is monoclonal antibody 4H4.
On the other hand, the present invention also provides a kind of isolated epitope peptides, by 6-15 company of HPV L1 albumen Continuous amino acid residue composition, and include the 303-308 amino acids residue of HPV L1 albumen.In a preferred embodiment party In case, the HPV L1 albumen is HPV16 L1 albumen.In a preferred embodiment, the of the HPV L1 albumen 303-308 amino acids residue is as shown in SEQ ID NO:58.
In a preferred embodiment, epitope peptide of the invention by L1 albumen not more than 15 continuous amino acids Residue composition, for example, it is residual by 15,14,13,12,11,10,9,8,7 or 6 continuous amino acids Base composition.For example, epitope peptide of the invention, which has, is selected from amino acid sequence shown in SEQ ID NO: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 epitope peptide of the invention be merged with carrier protein, to promote the presentation and enhancing epitope of epitope The immunogenicity of peptide.Therefore, the present invention also provides a kind of recombinant proteins, and it includes isolated epitope peptides and carrier of the invention Albumen, and not naturally occurring albumen or its segment.In the recombinant protein, the epitope peptide be can connect to carrier The N-terminal or C-terminal of albumen are inserted into the inside of carrier protein, or the partial amino-acid series of replacement 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, can also for example, it can be generated by gene engineering method (recombinant technique) To be generated by chemical synthesis process.
On the other hand, the present invention also provides a kind of isolated nucleic acid molecules, and it includes encode epitope of the 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 Isolated nucleic acid molecules.Carrier of the invention can be cloning vector, be also possible to 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 of the nucleic acid molecules or carrier comprising separating as described above is additionally provided.This Class host cell includes but is 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.).Cell of the invention is also It can be cell line, such as 293T cell.
On the other hand, the method for preparing recombinant protein of the invention is additionally provided comprising, under suitable conditions Host cell as described above is cultivated, and recycles recombinant protein of the 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, so that it has especially significant advantage in terms of detection and diagnosis.
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 The HPV infection or disease relevant to HPV infection (such as cervical carcinoma) of prevention or treatment subject have especially significant advantage.
Embodiment of the present invention is described in detail below in conjunction with drawings and examples, but those skilled in the art Member it will be understood that, following drawings and embodiment are merely to illustrate the present invention, rather than the restriction to 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.
Detailed description of the invention
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:HPV6 L1 albumen；Swimming lane 2:HPV11 L1 egg It is white；Swimming lane 3:HPV16 L1 albumen；Swimming lane 4:HPV18 L1 albumen；Swimming lane 5:HPV31 L1 albumen；Swimming lane 6:HPV33 L1 egg It is white；Swimming lane 7:EV71 albumen；Swimming lane 8:CRM197 albumen；Swimming lane 9:HEV495 albumen；Swimming lane 10:HPV35 L1 albumen；Swimming lane 11:HPV45 L1 albumen；Swimming lane 12:HPV52 L1 albumen；Swimming lane 13:HPV58 L1 albumen；Swimming lane 14:HPV59 L1 albumen； Swimming lane 15:EV71 albumen；Swimming lane 16:CRM197 albumen；Swimming lane 17:HEV495 albumen.Wherein, the protein sample of each swimming lane is dense Degree is 5 μ g/ml, and the concentration of used antibody 4B3 is 10 μ g/ml.The results show that the HPV L1 for the 11 kinds of types tested With 4B3 specific reaction can occur for albumen, and three kinds of unrelated proteins cannot be reacted with 4B3.This illustrates that monoclonal antibody 4B3 can wide spectrum And the L1 albumen of the HPV of a variety of types of specific binding.
Fig. 2 shows the testing result of western blot analysis, and which show 10 subclone peptides of HPV16 L1 albumen (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 HPV16 L1 albumen Peptide fragment HPV16L1F can be reacted with 4B3 with HPV16L1G, and remaining peptide fragment cannot be reacted with 4B3.This shows monoclonal antibody 4B3 In conjunction with epitope be located at position peptide fragment HPV16L1F Chong Die with HPV16L1G (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 (concentration is 5 μ g/ml) is as follows:
1,8,15,22:HPV6 L1 albumen of swimming lane；
2,9,16,23:HPV11 L1 albumen of swimming lane；
3,10,17,24:HPV16 L1 albumen of swimming lane；
4,11,18,25:HPV18 L1 albumen of swimming lane；
5,12,19,26:HPV33 L1 albumen of swimming lane；
6,13,20,27:HPV52 L1 albumen of swimming lane；
7,14,21,28:HPV58 L1 albumen of swimming lane.
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 being able to detect that 11 kinds of types when the concentration of the monoclonal antibody 4B3 used is at least 0.1 μ g/ml 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:HPV6 L1 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:HPV11 L1 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:HPV16 L1 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:HPV18 L1 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:HPV31 L1 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:HPV33 L1 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:HPV45 L1 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:HPV52 L1 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:HPV58 L1 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.
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 detection using 4B3, the detectable limit of the other L1 albumen of different shaped is had differences, and in 0.4 μ g/ml-3.6 μ Between g/ml.
Fig. 5 is shown using monoclonal antibody 4B3, and by immunoblotting, detection 65 have 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 egg in tissue of patient sample White to react 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
Sequence 1 (SEQ ID NO:1): 471bp
ATGGACAGACTTACATCTTCATTCCTGCTGCTGATTGTCCCTGCATATGTCCTTTCCCAGGTTACTCTG AAAGAGTCTGGCCCTGGGATATTGCAGACCTCCCAGACCCTCAGTCTGACTTGTTCTTCCTCTGGGTTTTCACTGAA CACCTCTGGTATGGGTGTGAGCTGGATTCGTCAGCCTTCAGGACAGGGTCTGGAGTGGCTGGCACACACTTACTGGG ATGATGACAAGCGCTATAATCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGAAACCTCCAGAAACCAGGTACTT CTCAAGATCACCAGTGTTGACACTGCAGATACTGCCACATACTACTGTGCTCGATATTTCTACGATAGTAACTACGG AGGGATGGACTACTGGGGTCAAGGAACCTCTGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCGTCTATCCACT GGTCCCTGGAATCTCTA
Sequence 2 (SEQ ID NO:2): 157aa
MDRLTSSFLLLIVPAYVLSQVTLKESGPGILQTSQTLSLTCSSSGFSLNTSGMGVSWIRQPSGQGLEWL AHTYWDDDKRYNPSLKSRLTISKETSRNQVLLKITSVDTADTATYYCARYFYDSNYGGMDYWGQGTSVTVSSAKTTP PSSIHWSLESL
Sequence 3 (SEQ ID NO:3): 387bp
ATGGTGTCCACAGCTCAGTTCCTTGGGATCTTGTTGCTCTGGTTTCCAGGTATCAGATGTGACATCACG ATGACCCAGTCTCCATCCTCCATGTATGCATCGCTGGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACAT TAAAAGTTATTTAAGCTGGTACCAACAGAAACCAAGGAAATCTCCTAAGACCCTGATCTATTATGCAACAAGGTTGT CAGATGGGGTCCCATCAAGATTCAGTGGCAGTGGATCTGGCCAAGATTATTCTCTAACCATCAGCAGCCTGGAGTCT GACGATACAGCAACTTATTACTGTCTACAACATTATGAGAGCCCGCTCACGTTCGGTCCTGGGACCAAGCTGGAAAT AAAACGGATC
Sequence 4 (SEQ ID NO:4): 129aa
MVSTAQFLGILLLWFPGIRCDITMTQSPSSMYASLGERVTITCKASQDIKSYLSWYQQKPRKSPKTLIY YATRLSDGVPSRFSGSGSGQDYSLTISSLESDDTATYYCLQHYESPLTFGPGTKLEIKRI
Sequence 5 (SEQ ID NO:5): 426bp
ATGAAATGCAGCTGGGTTATGTTCTTCCTGATGGCAGTGGTTACAGGGGTCAATTCAGAGGTTCAGCTG CAGCAGTCTGGGGCAGACCTTGTGAAGCCAGGGGCCTCAGTCAAGCTGTCCTGCACAGCTTCTGGCTTCAACATTAG AGACACCTTTATGCAGTGGGTGAAGCAGAGGCCTGAACAGGGCCCGGAGTACATTGGAAGGATTGATCCTGCAAATG GCTATGTTGAATATGGCCCGAAGTTCCAGGACAAGGCCACAATAACGGCAGACAAATCCTCCAACACAGCCTACCTT CAACTCAGCAGCCTGACATCTGAGGACACTGCCGTCTACTTCTGTAGTTCCTTCCATGGTCAACCTTTTGCTTATTG GGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCA
Sequence 6 (SEQ ID NO:6): 142aa
MKCSWVMFFLMAVVTGVNSEVQLQQSGADLVKPGASVKLSCTASGFNIRDTFMQWVKQRPEQGPEYIGR IDPANGYVEYGPKFQDKATITADKSSNTAYLQLSSLTSEDTAVYFCSSFHGQPFAYWGQGTLVTVSAAKTTPP
Sequence 7 (SEQ ID NO:7): 417bp
ATGGGCTTCAAGATGAAGTCACAGTTTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTATTAAA GGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTGGGAGACAGGGTCAACATCACCTGCATGGC CAGTCAGGATGTGGGTTCTGCTGTTGCCTGGTATCAACAGAAACCCGGACAATCTCCTAAATTACTGATTTATTGGG CATCCTCCCGGCACATTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAAC AATGTGCAGTCTGAAGACTTGGCAGATTATTTCTGTCAACAATATAGCACCTATACGTTCGGAGGGGGGACCAAGTT GGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATC
Sequence 8 (SEQ ID NO:8): 139aa
MGFKMKSQFQVFVYMLLWLSGIKGDIVMTQSHKFMSTSVGDRVNITCMASQDVGSAVAWYQQKPGQSPK LLIYWASSRHIGVPDRFTGSGSGTDFTLTINNVQSEDLADYFCQQYSTYTFGGGTKLEIKRADAAPTVSI
Sequence 9 (SEQ ID NO:9): 459bp
ATGAGAGTGCTGATTCTTTTGTGGCTGTTCACAGCCTTTCCTGGTGTCGTGTCTGATGTGGCGCTTCAG GAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCGCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAG TGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTTCATTACCAACAGTGATA CCACTAGCTACAATCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACGCATCCAAGAACCAGTTCTTCCTACAG TTGAGTTCTGTGACTACTGAGGACACAGCCACATATTATTGTGCAAGGTCCTCGGCCCCTCATTACTACGGCCTTTA CTGGGGCCAGGGGACTCTGGTCGCTGTCTCTGCAGCCAAAACGACACCCCCACCGTCTATCCACTGGTCCCTGGAAT CTCTA
Sequence 10 (SEQ ID NO:10): 153aa
MRVLILLWLFTAFPGVVSDVALQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGF ITNSDTTSYNPSLKSRISITRDASKNQFFLQLSSVTTEDTATYYCARSSAPHYYGLYWGQGTLVAVSAAKTTPPPSI HWSLESL
Sequence 11 (SEQ ID NO:11): 435bp
ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATTCTGTCCAGAGGACAA ATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGTTC AAGTGTAAATTACATGTATTGGTACCAGCAGAAGCCAGGATCTTCCCCCAGACTCCTGATTTATGACACATCCCGCC TGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCCAAATGGAG GCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGA AATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAATCTC
Sequence 12 (SEQ ID NO:12): 145aa
MDFQVQIFSFLLISASVILSRGQIVLTQSPAIMSASPGEKVTMTCSASSSVNYMYWYQQKPGSSPRLL IYDTSRLASGVPVRFSGSGSGTSYSLTI SQMEAEDAATYYCQQWSSYPYTFGGGTKLEIKRADAAPTVSIFPPSS NL
Sequence 13 (SEQ ID NO:13): 459bp
ATGAAATGCACCTGGGTTATTCTCTTTTTGATAGCAACAGCAACAGGTGTCCACTCCCAGGTCCAACTG CAGCAGTCTGGGGCTGAACTGGTGAACCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCCTCTGGCTACACCTTCAC CGACTACTATATATTTTGGGTGAAGCAGAGGCCTGGACTAGGCCTTGAGTGGATTGGAGAGATTAATCCTAACACTG GTGGTACTACCTTCAATGAGAGGTTCAAGGGCAAGGCCACACTTACTGTAGACAAATCCTCCAGTACAACATACATC CAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTACATCCTTTTACTACGGTAGTCCCTTTGACCA CTGGGGCCAAGGCACCACTCTCACAGTCTCCTCTGCCAAAACGACACCCCCATCGTTTATCCCTTGGCCCCTGGAAT CTCTA
Sequence 14 (SEQ ID NO:14): 153aa
MKCTWVILFLIATATGVHSQVQLQQSGAELVNPGASVKLSCKASGYTFTDYYIFWVKQRPGLGLEWIGE INPNTGGTTFNERFKGKATLTVDKSSSTTYIQLSSLTSEDSAVYYCTSFYYGSPFDHWGQGTTLTVSSAKTTPPSFI PWPLESL
Sequence 15 (SEQ ID NO:15): 435bp
ATGGATTTTCAAGTGCAGATTATCAGCTTCCTGCTAATCAGTGCCTCAGTCATGCTGTCCAGAGGACAA GTTGTTCTCATCCAGTCTCCAGCAATCATGTCTGTATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTTCCAGTTC AAGTATAGATTACATTCACTGGTACCAGCAGAAGCCAGGCACCTCCCCCAACAGATGGATTTATGACACATCCAAAT TGCCTTCTGGAGTCCCTGCTCGCTTCAGTAGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGTAGCATGGAG GCTGAGGATGCTGCCACTTATTACTGCCATCAGCGGAATAATTACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGA GCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAATCTC
Sequence 16 (SEQ ID NO:16): 145aa
MDFQVQI ISFLLISASVMLSRGQVVLIQSPAIMSVSPGEKVTMTCSSSSSIDYIHWYQQKPGTSPNR WIYDTSKLPSGVPARFSSSGSGTSYSLTI SSMEAEDAATYYCHQRNNYPLTFGAGTKLELKRADAAPTVSIFPPS SNL
Sequence 17 (SEQ ID NO:17): 10aa
GFSLNTSGMG
Sequence 18 (SEQ ID NO:18): 7aa
TYWDDDK
Sequence 19 (SEQ ID NO:19): 14aa
ARYFYDSNYGGMDY
Sequence 20 (SEQ ID NO:20): 6aa
QDIKSY
Sequence 21 (SEQ ID NO:21): 3aa
YAT
Sequence 22 (SEQ ID NO:22): 9aa
LQHYESPLT
Sequence 23 (SEQ ID NO:23): 8aa
GFNIRDTF
Sequence 24 (SEQ ID NO:24): 8aa
IDPANGYV
Sequence 25 (SEQ ID NO:25): 10aa
SSFHGQPFAY
Sequence 26 (SEQ ID NO:26): 6aa
QDVGSA
Sequence 27 (SEQ ID NO:27): 3aa
WAS
Sequence 28 (SEQ ID NO:28): 7aa
QQYSTYT
Sequence 29 (SEQ ID NO:29): 9aa
GYSITSDYA
Sequence 30 (SEQ ID NO:30): 7aa
ITNSDTT
Sequence 31 (SEQ ID NO:31): 12aa
ARSSAPHYYGLY
Sequence 32 (SEQ ID NO:32): 5aa
SSVNY
Sequence 33 (SEQ ID NO:33): 3aa
DTS
Sequence 34 (SEQ ID NO:34): 9aa
QQWSSYPYT
Sequence 35 (SEQ ID NO:35): 8aa
GYTFTDYY
Sequence 36 (SEQ ID NO:36): 8aa
INPNTGGT
Sequence 37 (SEQ ID NO:37): 11aa
TSFYYGSPFDH
Sequence 38 (SEQ ID NO:38): 5aa
SSIDY
Sequence 39 (SEQ ID NO:39): 3aa
DTS
Sequence 40 (SEQ ID NO:40): 9aa
HQRNNYPLT
Sequence 41 (SEQ ID NO:41): 15aa
AQIFNKPYWLQRAQG
Sequence 42 (SEQ ID NO:42): 14aa
QIFNKPYWLQRAQG
Sequence 43 (SEQ ID NO:43): 13aa
IFNKPYWLQRAQG
Sequence 44 (SEQ ID NO:44): 12aa
FNKPYWLQRAQG
Sequence 45 (SEQ ID NO:45): 11aa
NKPYWLQRAQG
Sequence 46 (SEQ ID NO:46): 10aa
KPYWLQRAQG
Sequence 47 (SEQ ID NO:47): 9aa
PYWLQRAQG
Sequence 48 (SEQ ID NO:48): 8aa
YWLQRAQG
Sequence 49 (SEQ ID NO:49): 7aa
WLQRAQG
Sequence 50 (SEQ ID NO:50): 14aa
AQIFNKPYWLQRAQ
Sequence 51 (SEQ ID NO:51): 13aa
AQIFNKPYWLQRA
Sequence 52 (SEQ ID NO:52): 12aa
AQIFNKPYWLQR
Sequence 53 (SEQ ID NO:53): 11aa
AQIFNKPYWLQ
Sequence 54 (SEQ ID NO:54): 10aa
AQIFNKPYWL
Sequence 55 (SEQ ID NO:55): 9aa
AQIFNKPYW
Sequence 56 (SEQ ID NO:56): 8aa
AQIFNKPY
Sequence 57 (SEQ ID NO:57): 7aa
AQIFNKP
Sequence 58 (SEQ ID NO:58): 6aa
IFNKPY
Sequence 59 (SEQ ID NO: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, deposit number CCTCC-C201264, preservation time are on May 31st, 2012；
Hybridoma cell strain 4H4, deposit number CCTCC-C201265, preservation time are on May 31st, 2012；
Hybridoma cell strain 12B9, deposit number CCTCC-C201266, preservation time are on May 31st, 2012；
Hybridoma cell strain 13A10, deposit number CCTCC-C201267, preservation time are on May 31st, 2012.
Specific embodiment
It is intended to illustrate the present invention embodiment (rather than limiting the invention) referring now to following and describes the present invention.
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 or commercially available reagent of this field.As known to those skilled in the art, 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 the anti-HPVL1 protein monoclonal antibody of embodiment 1.
This experiment passes through the HPV VLP cross immunity mouse with multiple types, by the splenocyte and bone of immunized mouse Myeloma cells fusion, and is screened, and obtains having the HPV VLP of multiple types the thin of reactive antibody and secretory antibody 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 method.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 with HPV VLP and reacts titre.When titre reaches To 106When above, take Mouse spleen cells for merging.72hr carries out booster immunization again before merging: through tail vein injection 1 Secondary, 50 μ l/ are only.Prepare 60 blocks of fusion plates.
Fusion: the spleen cell of highest 3 mouse of serum titer is taken, is blended with murine myeloma cell.First by spleen Dirty grinding, obtains splenocyte suspension；Then by its SP2/0 mouse bone marrow cells in logarithmic growth phase with low ten times of cell number Oncocyte mixing, and 1min is acted on 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 medium 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 antibody secreted by cell strain stable bond HPV VLP and can neutralize Until HPV pseudovirus.
The selection result: the cell strain of four plants of secrete monoclonal antibodies: 4B3,13A10,12B9,4H4 is obtained.
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 Flask is 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 first uses 50% ammonium sulfate precipitation to handle, and then dialyses to PBS, pH7.2, is then carried out with DEAE column HPLC purifying, finally obtains purified monoclonal antibody.SDS-PAGE detection display, the purity of purified monoclonal antibody Reach 95% or more.
The reactive ELISA of the HPV VLP of monoclonal antibody and each type is 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, then obtained with 3 times of concentration gradient dilution additions Monoclonal antibody (maximum concentration 1mg/ml).The combination for detecting the HPV VLP of each monoclonal antibody and each type 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 in conjunction 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 Have 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 reacts；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, HPV 58 VLP react.
The response intensity of the HPV VLP of table 2:4 kind monoclonal antibody and 11 types
The reactive Western blotting (WB) of the HPV L1 albumen of monoclonal antibody and 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 electrophoresis: 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 electrophoresis.
Transferring film: will be on the albumen electrotransfer to nitrocellulose filter on SDS- polyacrylamide gel.
Closing: nitrocellulose filter is immersed in 5% defatted milk, closes 60min.
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: using 20mM Tirs-HCl, pH8.0, and 0.5M NaCl, 0.05%Tween20 solution washs nitrocellulose Film 5 times, each 3min.
Enzyme: 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: using 20mM Tirs-HCl, pH8.0, and 0.5M NaCl, 0.05%Tween20 solution washs nitrocellulose Film 5 times, each 3min.
Colour developing: each 50ul of color developing agent A, B liquid (color developing agent A liquid: 13.4g/L Na is added in every hole2HPO4.12H2O+4.2g/L Citric acid .H2O+0.3g/L carbamide peroxide；Color developing agent B liquid: 0.2mM/L tetramethyl benzidine (TMB)+20mM/L dimethyl methyl Amide；Similarly hereinafter), develop the color 10min.
Antibody 4B3 and the reactive WB testing result 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 result is 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
It, can be using the method for cDNA clones to its epitope since the antibody 4B3 epitope identified is linear epitope It is positioned.
Firstly, cDNA clones are expressed in Escherichia coli by HPV16 L1 albumen, 10 subclone peptides, each peptide are prepared altogether 65aa, and (aa indicates amino acid, and the n-th bit amino is indicated when being placed in front of 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 HPV16 L1 The aa 1-65, the aa 51-115, HPV16L1C that HPV16L1B corresponds to HPV16 L1 albumen of albumen correspond to HPV16 L1 egg White aa 101-165, and so on.
This 10 subclone peptides and the WB reactivity of monoclonal antibody 4B3 are detected, as a result as 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 overlapping Place, that is, positioned at the aa301-315 of HPV16 L1 albumen, sequence is as shown in SEQ ID NO:41.
Further, 17 segment polypeptides have been synthesized for the design of this 15 amino acid (SEQ ID NO:41), be respectively designated as L1FG1-L1FG17, amino acid sequence are respectively SEQ ID NO:41-57 (synthesis of Shanghai Sheng Gong biotech firm).Then, lead to Cross the combination situation that competitive ELISA tests and analyzes these polypeptides (L1FG1-L1FG17) and monoclonal antibody 4B3.Specific step 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: will be in HPV16L1 protein adsorption to 8K-96 orifice plate.
Sample-adding: 19 samples after incubation are added to respectively in the hole for being coated with HPV16 L1.
It is incubated for: sealing 96 orifice plates with sealing plate film, react 30min in 37 DEG C of biochemical cultivation cases.
Washing: carefully taking sealing plate film off, is washed 5 times with board-washing machine washing, and last time button as far as possible is dry.
Enzyme: 100 μ l GAM-AP are added in every hole.
It is incubated for: sealing 96 orifice plates with sealing plate film, react 30min in 37 DEG C of biochemical cultivation cases.
Washing: carefully taking sealing plate film off, is washed 5 times with board-washing machine washing, and last time button as far 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 is added in every 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, and sequence is SEQ ID NO:58, corresponds to HPV16L1 egg The aa303-308 of white (SEQ ID NO:59).
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 software The sequence of L1 albumen is compared, and analysis corresponds to the sequence of the aa303-308 of HPV16 L1 albumen (SEQ ID NO:59) Homology.It is searched for and is screened, sequence of the HPV in the section of 121 types is obtained from ncbi database.To these sequences It is compared, and for statistical analysis to the conservative of each amino acid.
It analyzes shown in result Table A.The results show that the aa303-308 of HPV16 L1 albumen in 121 HPV types very It is conservative, wherein the 303rd, 304,305, the homologys of 307 amino acids 85% or more (the 304th, 305,307 amino acids 95%) homology has been even more than；306th, the difference of 308 amino acids is relatively large, but homology still reaches 50% or more. This explanation, the epitope that 4B3 is identified are conserved epitope, are extremely conservative section in the HPV of multiple types, and therefore structure At the wide spectrum identification of monoclonal antibody 4B3 and in conjunction with 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 Its expression for being used to detect 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 detection pole of the test monoclonal antibody 4B3 to the HPV L1 of 11 kinds of types Limit；Finally, having detected 63 precancerous lesions of uterine cervixs of HPV DNA test positive with monoclonal antibody 4B3 by western blot analysis 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 electrophoresis: 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 electrophoresis.
Transferring film: will be on the albumen electrotransfer to nitrocellulose filter on SDS- polyacrylamide gel.
Closing: nitrocellulose filter is immersed in 5% defatted milk, closes 60min.
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: using 20mM Tirs-HCl, pH8.0, and 0.5M NaCl, 0.05%Tween20 solution washs nitrocellulose Film 5 times, each 3min.
It is enzyme: to add GAM-AP on nitrocellulose filter, react 1 hour.
Washing: using 20mM Tirs-HCl, pH8.0, and 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 is added in every hole.
As a result as 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 egg Bai Junneng reacts.Therefore, the monoclonal antibody 4B3 of such as 1 μ g/ml can be used to carry out WB detection.
WB detectable limit of the 4B3 to the HPV L1 albumen of each type
SDS- polyacrylamide gel electrophoresis: the L1 albumen of HPV6,11,16,18,31,33,45,52,58 is diluted respectively SDS- polyacrylamide gel electrophoresis is carried out for 7 concentration: 100,33,11,3.6,1.2,0.4,0.1 μ g/ml.
Transferring film: will be on the albumen electrotransfer to nitrocellulose filter on SDS- polyacrylamide gel.
Closing: nitrocellulose filter is immersed in 5% defatted milk, closes 60min.
Add antibody: antibody 4B3 be diluted to 1 μ g/ml with 5% defatted milk, then with nitrocellulose film reaction 1 hour.
Washing: using 20mM Tirs-HCl, pH8.0, and 0.5M NaCl, 0.05%Tween20 solution washs nitrocellulose Film 5 times, each 3min.
It is enzyme: to add GAM-AP on nitrocellulose filter, react 1 hour.
Washing: using 20mM Tirs-HCl, pH8.0, and 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 is added in every hole.
As a result as shown in Fig. 4 and table 3.The result shows that when carrying out WB detection 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.
The detectable limit of the L1 albumen of table 3:HPV6,11,16,18,31,33,45,52,58
The horizontal detection of HPV L1 albumen in cervical exfoliated cell
Sample acquisition: the 65 of Zhongshan Hospital Xiamen University's OPD of Obs & Gyn (in June, 2010 in September, 2011) are come from Example patient, the DNA detection of their cervical exfoliated cell are the high-risk HPV positive.Cervical exfoliated cell is acquired respectively, is saved In PBS solution.
Sample treatment: taking 100 μ L of cervical exfoliated cell sample, and 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) glycerol, 5% (v/v) beta -mercaptoethanol, Similarly hereinafter), it mixes and in 80 DEG C of water-bath 10min.
SDS- polyacrylamide gel electrophoresis: processed sample is subjected to SDS- polyacrylamide gel electrophoresis.
Transferring film: will be on the albumen electrotransfer to nitrocellulose filter on SDS- polyacrylamide gel.
Closing: nitrocellulose filter is immersed in 5% defatted milk, closes 60min.
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: using 20mM Tirs-HCl, pH8.0, and 0.5M NaCl, 0.05%Tween20 solution washs nitrocellulose Film 5 times, each 3min.
It is enzyme: to add GAM-AP on nitrocellulose filter, react 1 hour.
Washing: using 20mM Tirs-HCl, pH8.0, and 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 is added in every hole.
Fig. 5 is shown using 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 using the HPV L1 in 4B3 detection patient samples.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.
The recall rate of table 4:HPV L1 albumen and the relationship of patient's pathology
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) 2003 grade scales formulated).
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, referring 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.
Firstly, then 2 times of concentration gradient doubling dilutions (maximum concentration 1mg/ml) of 13A10 antibody are directed to each dilution Degree, takes 50 μ l respectively, it is mixed in 96 orifice plates with the HPV16 pseudovirus (MOI=0.1) of the suitable concentration of 50 μ l, and 4 DEG C be incubated for a hour.Using the mixed liquor of pseudovirus and PBS as negative control.Then each mixed liquor is separately added into preparatory paving There is in 96 porocyte plates of 293FT cell (every hole about 1.5 × 104293FT cell), 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 company, 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 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 is living with cross-neutralization to two types of HPV33, HPV58 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 the reactivity of wide spectrum to HPV L1 albumen and VLP, but also have the HPV's for being directed at least two type Cross-neutralization activity.
Table 5: neutralization activity of each monoclonal antibody to the HPV pseudovirus of 11 types
The affinity analysis of 5. monoclonal antibody of embodiment
This experiment uses BIACORE3000 biosensor (GE company, the U.S.), analyzes antigen-antibody and combines and dissociate Dynamics, calculate the binding constant k of monoclonal antibody 13A10 Yu various HPV VLPa, dissociation constant kdWith affinity KD, To reflect the power of the combination degree of the antibody and various HPV VLP.
Firstly, with the Acetic acid-sodium acetate buffer of pH 5.5 by sheep anti mouse mostly it is anti-(GAM) (be purchased from U.S. KPL company, under It is diluted to 40 μ g/ml together).According to the specification (Biacore3000 biosensor, GE company, the U.S.) of manufacturer, by GAM idol 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.Meanwhile with HBS-EP buffer 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 with the glycine-HCI buffer 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 software.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 analysis of the antibody 13A10 to the HPV VLP of multiple types
6. monoclonal antibody of embodiment is to serum-antigen binding blocking experiment
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 lower than 20%, not hinder It is disconnected.It on the other hand 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 16 capsids 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 acquisition of HPV VLP respectively, HPV16/18 bivalent vaccine inoculator's Serum and the serum of natural HPV infection person carry out blocking experiment, to investigate antibody in corresponding HPV VLP, HPV16/18 vaccine Whether the epitope identified in antigen and natural HPV is Dominant Epitopes, and specific experiment process is as follows.
The preparation of ELISA Plate:
The VLP for being coated with HPV16,31,33,58 respectively on 8K-96 orifice plate (8 rows × 12 column) is (slow using 20mM phosphate Fliud flushing (PB, pH7.4) and 0.3M NaCl solution 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, it is added confining liquid (casein+sucrose), and in 37 DEG C of closing 2hr.Again by plate Secondary button dry doubling vacuum is drained, and is obtained in ELISA Plate (hole 8x12).
The preparation of other reagents needed for blocking experiment:
Enzyme marking reagent: with more anti-(GAM) (being purchased from U.S. KPL company) of HRP label sheep anti mouse 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 peroxide；
Color developing agent B liquid: 3,3 ', 5,5 '-tetramethyl benzidine of 0.2mM/L (TMB)+20mM/L dimethylformamide；
Terminate liquid: 2M sulfuric acid；
Concentrated cleaning solution: 20x PBST.
Detection process:
With liquid: 50ml concentrated cleaning solution (20 ×) is diluted to 1000ml with distilled water or deionized water, it is spare.
Number: corresponding to microwell plate for sample and sequentially 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 every row of ELISA Plate is added, then by the concentration dilution of antibody 3 Times the second hole is added, and successively doubling dilution (that is, the concentration in previous hole is 3 times of latter hole) backward, obtains 11 dilutions altogether Degree (3 times of serial dilutions, maximum concentration are 100 μ g/ml).The dilution for being free of antibody is added in last hole of every row.
It is incubated for: using sealed membrane sealing plate, place it in biochemical cultivation case, 37 DEG C of incubation 60min.
Washing: carefully taking sealing plate film off, is washed 5 times with board-washing machine washing, and last time button as far as possible is dry.
The pretreatment of the serum or antibody that are blocked and sample-adding:
Sample is serum sample: firstly, serum sample and corresponding type by ELISA detection for blocking experiment The combination situation of VLP.Then, dilute serum sample according to testing result, makes it in conjunction with the ELISA of the VLP of corresponding type OD value is 1 or so.It is used for the diluted serum sample to carry out following test.
It is incubated for: using sealed membrane sealing plate, place it in biochemical cultivation case, 37 DEG C of incubation 60min.
Washing: carefully taking sealing plate film off, is washed 5 times with board-washing machine washing, and last time button as far as possible is dry.
It is enzyme: 100 μ l of enzyme marking reagent is added in corresponding aperture respectively.
It is incubated for: using sealed membrane sealing plate, place it in biochemical cultivation case, 37 DEG C of incubation 45min.
Washing: carefully taking sealing plate film off, is washed 5 times with board-washing machine washing, and last time button as far 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 is added in every hole.
Measurement: every hole adds terminate liquid 1 to drip (50 μ l), then with microplate reader with Single wavelength 450nm (need to set blank control wells) Or dual wavelength 450nm/630nm measures each hole OD value.
Sample is the specific antibody for resisting each type: every plant of specific antibody marked with HRP, it then directly will be through marking The antibody of note dilutes 1000 times, and in adding hole.
It is incubated for: using sealed membrane sealing plate, place it in biochemical cultivation case, 37 DEG C of incubation 60min.
Washing: carefully taking sealing plate film off, is washed 5 times with board-washing machine washing, and last time button as far 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 is added in every hole.
Measurement: every hole adds terminate liquid 1 to drip (50 μ l), then with microplate reader with Single wavelength 450nm (need to set blank control wells) Or dual wavelength 450nm/630nm measures each hole OD value.
Result judgement:
A) normal range (NR) of negative control: under normal circumstances, value≤0.1 OD of negative control hole (if negative control hole OD value is greater than 0.1, then should give up；If the OD value of all negative control holes is both greater than 0.1, experiment should be repeated；If negative control The OD value in hole is then calculated by 0.03 less than 0.03).
B) calculating of critical value (CUTOFF): the mean value+0.15 of negative control hole OD value.
C) judgement of single hole blocking rate: the OD value in every hole is compared with the OD value in the hole for not adding monoclonal antibody to be measured, and will The blocking rate in every hole calculates are as follows:
Blocking rate=(hole 1- OD value)/1 × 100%
D) calculating of antibody blocking rate: using monoclonal antibody concentration used in each hole as abscissa, it is with the blocking rate in the hole Ordinate maps and is fitted to parabola.Blocking rate when calculating antibody concentration is 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 obtain the rabbit anteserum (each 2 parts) with the immune acquisition in HPV16,31 or 33,12B9 to HPV33 or 58 is immune Rabbit anteserum (each 2 parts) blocking rate.Testing result is as shown in table 7- table 9.
, the results show that antibody 13A10 is higher than 50% to the blocking rate of the rabbit anteserum of 2 parts of HPV58, this shows antibody for these 13A10 is the advantage antibody of HPV58 VLP immune serum, and the epitope identified is the Dominant Epitopes of HPV58 VLP；Antibody 13A10 is below 50% to the blocking rate of the rabbit anteserum of HPV16,31,33, this show antibody 13A10 be not HPV16,31, The advantage antibody of 33VLP immune serum.Similarly, antibody 4H4 and 12B9 is also not the VLP immune serum of HPV16,31,33,58 Advantage antibody.
Blocking of the table 7:13A10 to the rabbit anteserum of HPV16,31,33,58
Blocking of the table 8:4H4 to HPV33,58 rabbit anteserum
Blocking of the table 9:12B9 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- table 11.
These the results show that antibody 13A10 is higher than 50% to the blocking rate of the serum of 14 parts of HPV58 natural infection persons, 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 also not the natural infection person of HPV16,31,33 Serum advantage antibody.
Blocking of the table 10:13A10 to the serum of the natural infection person of HPV16,58 or 33
Blocking of the table 11:4H4 to the serum of the natural infection person of HPV16,58 or 33
The light chain gene of 7. monoclonal antibody of embodiment and the separation of heavy chain gene variable region
Half adhere-wall culture 107A hybridoma blows afloat adherent cell with blowpipe and is allowed to suspend, and is transferred into new 4ml centrifuge tube in.It is centrifuged 3min with 1500rpm, collects the cell of precipitating, and be resuspended in the 100 sterile PBS of μ l (pH7.45) in.Cell suspension is transferred in a new 1.5ml centrifuge tube, 800 μ l Trizol of addition (Roche, Germany it), and is gently mixed by inversion, stands 10min.Then, 200 μ l chloroforms are added and acutely vibrate 15s, stand 10min. Later, it is centrifuged 15min, and shifts supernatant liquid into a new 1.5ml centrifuge tube with 4 DEG C, 12000rpm, be added isometric Isopropanol mixes and stands 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 Precipitating is dissolved in 70 μ l DEPC H2In O, and it is distributed into two pipes.Every pipe is separately added into 1 μ l reverse transcription primer, 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'-CggT 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 (the raw work in Shanghai) is 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 transcription at cDNA.
Using polymerase chain reaction (PCR) method separation antibody gene variable region, the Ig- according to Novagen company is used The primer sets (table 12) of Prime kit synthesis and two other downstream primer MVJkR and MVDJhR (Shanghai Bo Ya companies Synthesis), wherein MVJkR is the downstream primer expanded for light-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 condition are as follows: 94 DEG C of 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 carrier, Then (Shanghai Bo Ya company) is sequenced.Blas t comparison is carried out to sequencing sequence, to determine the nucleotide of antibody variable region Sequence, 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 has been determined.Table 12 shows the sequence of used upstream primer.Table 13 show the sequence number of the nucleotide sequence of the heavy chain of 4 plants of monoclonal antibodies and light chain variable region and amino acid sequence.Table 14 show according to Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) described in method determine 4 plants of monoclonals The cdr amino acid sequence of antibody.
Table 12: for expanding the sequence of the upstream primer of monoclonal antibody variable region gene
The sequence number of the variable region of 13:4 plants of monoclonal antibodies of table
The cdr amino acid sequence of 14:4 plants of monoclonal antibodies of table
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, to the wide spectrum of HPV L1 albumen reactivity, and/or to the cross-neutralization activity of the HPV of at least two type).
Although a specific embodiment of the invention has obtained detailed description, those skilled in the art will appreciate that root According to all introductions having disclosed, details can be carry out various modifications and be changed, and these change in guarantor of the invention Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (32)

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:
Comprising amino acid sequence be SEQ ID NO:17-19 CDR VH, and comprising amino acid sequence be SEQ ID NO:20- The VL of 22 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:
The VH as shown in the SEQ ID NO:2 and VL as shown in SEQ ID NO:4.
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 can Specific binding selected from following HPV L1 albumen and VLP:HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52, HPV58 and HPV59.
6. 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.
7. the monoclonal antibody of any one of claim 1-2 or its antigen-binding fragment, wherein the non-CDR region is anti-from people Body.
8. the monoclonal antibody of any one of claim 1-2 or its antigen-binding fragment, wherein the monoclonal antibody be by The monoclonal antibody that hybridoma cell strain 4B3 is generated, the hybridoma cell strain 4B3 are preserved in China typical culture collection The heart (CCTCC), and there is deposit number CCTCC-C201264.
9. isolated nucleic acid molecules, it includes the nucleic acid sequences for capableing of encoding antibody heavy variable region and being capable of encoding antibody light The nucleic acid sequence of variable region, wherein the antibody heavy chain variable region includes: amino acid sequence is the CDR of SEQ ID NO:17-19, The antibody's light chain variable region includes: amino acid sequence is the CDR of SEQ ID NO:20-22.
10. the isolated nucleic acid molecules of claim 9, wherein the antibody heavy chain variable region has shown in SEQ ID NO:2 Amino acid sequence.
11. the isolated nucleic acid molecules of claim 9, wherein the nucleic acid sequence tool for capableing of encoding antibody heavy variable region There is nucleotide sequence shown in SEQ ID NO:1.
12. the isolated nucleic acid molecules of claim 9, wherein the antibody's light chain variable region has shown in SEQ ID NO:4 Amino acid sequence.
13. the isolated nucleic acid molecules of claim 9, wherein the nucleic acid sequence tool for capableing of encoding antibody light variable region There is nucleotide sequence shown in SEQ ID NO:3.
14. a kind of carrier, it includes the isolated nucleic acid molecules of any one of claim 9-13.
15. a kind of host cell, it includes the loads of the isolated nucleic acid molecules or claim 14 of any one of claim 9-13 Body.
16. preparing the monoclonal antibody of any one of claim 1-8 or the method for its antigen-binding fragment comprising, suitable Under conditions of cultivate the host cell of claim 15, and the monoclonal antibody or its antigen knot are recycled from cell culture Close segment.
17. hybridoma cell strain 4B3 is preserved in China typical culture collection center (CCTCC), and has deposit number CCTCC-C201264.
18. kit comprising the monoclonal antibody of any one of claim 1-8 or its antigen-binding fragment.
19. the kit of claim 18, wherein the monoclonal antibody or its antigen-binding fragment further include detectable mark Note.
20. the kit of claim 19, wherein the detectable label is selected from radioactive isotope, and luminescent substance is coloured Substance and enzyme.
21. the kit of claim 19, wherein it is described it is detectable label be.
22. the kit of claim 19, 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.
23. the kit of claim 22, wherein the detectable label is selected from radioactive isotope, luminescent substance is coloured Substance and enzyme.
24. the kit of claim 22, wherein it is described it is detectable label be.
25. the method for the presence for being used to detect HPV L1 albumen or VLP in the sample or its horizontal non-diagnostic purpose comprising Use the monoclonal antibody or its antigen-binding fragment of any one of claim 1-8.
26. the method for claim 25, wherein the monoclonal antibody or its antigen-binding fragment further include detectable mark Note.
27. the method for claim 26, wherein the detectable label is selected from radioactive isotope, luminescent substance, colored substance Matter and enzyme.
28. the method for claim 26, wherein it is described it is detectable label be.
29. the method for claim 25, wherein the method also includes being come using the secondary antibody for carrying detectable label Detect the monoclonal antibody or its antigen-binding fragment.
30. the method for claim 29, wherein the detectable label is selected from radioactive isotope, luminescent substance, colored substance Matter and enzyme.
31. the method for claim 29, wherein it is described it is detectable label be.
32. the purposes of the monoclonal antibody of any one of claim 1-8 or its antigen-binding fragment in reagent preparation box, described The presence or its level that kit is used to detect HPV L1 albumen or VLP in the sample, or for diagnosing whether subject infects HPV.
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Families Citing this family (21)

* Cited by examiner, † Cited by third party
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CN106460074A (en) * 2014-03-27 2017-02-22 亚利桑那州立大学董事会 HPV16 antibodies as diagnostic and prognostic biomarkers in pre-invasive and invasive disease
CN105987997B (en) * 2015-01-30 2017-12-29 江苏众红生物工程创药研究院有限公司 The fluorogenic quantitative detection test card of human tissue kallikrein 1
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CN108794623B (en) * 2018-07-04 2021-08-13 北京索莱宝科技有限公司 Monoclonal antibody for resisting HPV16E6 protein and application thereof
CN111116740B (en) * 2019-12-30 2022-05-31 中生方政生物技术股份有限公司 Monoclonal neutralizing antibody of HPV33L1 and application thereof
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CN111205364B (en) * 2020-02-26 2022-05-17 中生方政生物技术股份有限公司 Monoclonal neutralizing antibody against HPV31L1 and application thereof
CN111205365B (en) * 2020-02-26 2022-05-17 中生方政生物技术股份有限公司 Monoclonal neutralizing antibody against HPV59L1 and application thereof
CN112409487B (en) * 2020-11-27 2022-04-29 宁波赛珀生物技术有限公司 Anti-soluble transferrin receptor antibodies and uses thereof
CN113512109B (en) * 2021-03-09 2022-03-25 北京康乐卫士生物技术股份有限公司 Monoclonal neutralizing antibody against human papillomavirus 31 and application thereof
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CN114195886B (en) * 2021-11-12 2023-05-23 郑州大学 anti-HPV 39L1 protein monoclonal antibody, preparation and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995001374A1 (en) * 1993-07-01 1995-01-12 British Technology Group Limited Synthetic peptides of human papillomavirus
DE4332596A1 (en) * 1993-09-24 1995-03-30 Martin Josef Dr Sapp Monoclonal antibodies
CN101538319A (en) * 2007-12-06 2009-09-23 三峡大学 Polypeptide for detecting polytype human papilloma virus and application thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10101890A1 (en) * 2001-01-16 2002-08-01 Deutsches Krebsforsch HPV-specific peptides that block the binding of HPV to the host cell
CA3012796C (en) * 2010-02-16 2021-08-31 Osterreichische Akademie Der Wissenschaften Anti-hpv e7 antibodies

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995001374A1 (en) * 1993-07-01 1995-01-12 British Technology Group Limited Synthetic peptides of human papillomavirus
DE4332596A1 (en) * 1993-09-24 1995-03-30 Martin Josef Dr Sapp Monoclonal antibodies
CN101538319A (en) * 2007-12-06 2009-09-23 三峡大学 Polypeptide for detecting polytype human papilloma virus and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Identification of Two Cross-Neutralizing Linear Epitopes within the L1 Major Capsid Protein of Human Papillomaviruses.;Alba-Lucia Combita等;《JOURNAL OF VIROLOGY》;20020731;6480-6486 *

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