CN104031144A

CN104031144A - Antibody specifically binding to type 3, 4 hepatitis E viruses and application thereof

Info

Publication number: CN104031144A
Application number: CN201310069913.4A
Authority: CN
Inventors: 郑子峥; 唐自闽; 赵敏; 李晓静; 温桂平; 李婧娴; 张军; 夏宁邵
Original assignee: Xiamen University; Xiamen Innovax Biotech Co Ltd
Current assignee: Xiamen University; Xiamen Innovax Biotech Co Ltd
Priority date: 2013-03-05
Filing date: 2013-03-05
Publication date: 2014-09-10
Anticipated expiration: 2033-03-05
Also published as: CN104031144B

Abstract

The invention relates to hepatitis E virus antibodies, in particular to an antibody that specifically binds to type 3, 4 hepatitis E viruses and does not bind to or weakly binds to type 1 hepatitis virus. For instance, the antibody can be HEV-C3-1 or HEV-C3-2. The invention also relates to type 3, 4 hepatitis E viruses' antigen epitope specifically binding to the antibody. The invention also relates to a kit or detection reagent containing the antibody or antigen epitope, and application of the antibody or antigen epitope in preparation of kits or detection reagents and application in preparation of drugs for preventing or treating hepatitis E.

Description

Antibody of specific combination hepatitis E virus 3,4 types and uses thereof

Technical field

The present invention relates to hepatitis E virus antibody, being specifically related to can specific binding 3,4 type hepatitis E virus and be not combined with 1 Hepatitis virus or the antibody of weak binding.The invention still further relates to the epitope of 3, the 4 type hepatitis E viruss of being combined with described antibodies specific.The invention still further relates to the test kit that comprises described antibody or epitope or detection reagent, described antibody or epitope for the preparation of the purposes of test kit or detection reagent and for the preparation of the purposes of the medicine of prevention or treatment hepatitis E.

Background technology

There is the acute viral hepatitis outburst causing because of drinking water pollution in India's the 1950's New Delhi.It is believed that at that time it is by hepatitis A virus (Hepatitis A Virus, HAV) cause, retrospective study is for many years found: in patients serum, lack hepatitis A virus infection index, prompting has another kind can cause that the cause of disease of popular acute hepatitis exists, people are the viral hepatitis called after enteron aisle non-A non-B hepatitis of this unknown cause (enterically transmitted non-A non-B hepatitis, ET-NANBH).The Identification of etiology first of this virus (Balayan, M.S.et al., Intervirology, 1983,20 (1): 23-31) that to be nineteen eighty-three Balayan etc. obtain from a volunteer's research.Utilize immunoelectronmicroscopy this volunteer's acute phase ight soil and convalescence serum in find a kind of diameter 27nm(27～34nm) nonenveloped virus particle, and find that this virus can successfully infect cynomolgus monkey.Nineteen ninety Reyes etc. is hepatitis E (viral hepatitis type E by ET-NANBH definite designation, Hepatitis E, HE) (Reyes, G.R.et al., Science, 1990,247 (4948): 1335-1339), corresponding infective pathogen body is named as hepatitis E virus (viral hepatitis type E virus, Heptatitis E Virus, HEV).In recent years, researchist has found the evidence that HEV infects successively from the many animals such as pig, deer, monkey, and finds that animal HEV and human body HEV have gene similarity (Lu L, Li C et al., Rev Med Virol, 2006, the 16:5-36 of height; Qu Lichun, Guo Zhi scholar et al., animal and veterinary science, 2006,22 (8): 26-29).Family pig (the Meng with it that the first animal-origin HEV was in the news in 1997 and is located away from the U.S.; X.J.et al.; Proc Natl Acad Sci U S A, 1997,94 (18): 9860-9865); this has opened up the diverse visual field for the research of HEV; start since then nearly all using pig as meat product for application domestic animal country all pig, be separated to HEV (Meng, X.J., Curr Top Microbiol Immunol with it; 2003,278:185-216; Wilhelm, B.J.et al., Epidemiol Infect, 2011,139 (8): 1127-1144).Japanology personnel also find the mankind at the edible venison postoperative infection that contains HEV viral hepatitis type E.These evidences all demonstrate viral hepatitis type E be a kind of zoonosis (Tei S, Kitajima N et al., Lancet, 2003,362:371-373), human health is formed to huge threat.

The hepatitis E that hepatitis E virus (HEV) causes is similar to hepatitis A in clinical symptom, but case fatality rate is higher, and third trimester of pregnancy, case fatality rate can be up to 15-25%.All the time, viral hepatitis type E in outburst all over the world constantly.Within 1986～1988 years, in Xinjiang of China, once there is the viral hepatitis type E great outburst of accumulation morbidity more than 110,000 people, dead nearly 1000 people together, extremely serious social influence (Zhuan Hui, Liu Chong cypress et al., Peking University's journal (medicine) have been caused, 1992,24 (4): 281-284).Ministry of Health's announcement data shows, China's viral hepatitis type E morbidity in recent years increases rapidly, annual viral hepatitis type E morbidity report 6830 examples in 2002, within 2003, rise to 9655 examples, within 2004, rise to 16436 examples, become generally popular, endanger one of serious disease (http://www.moh.gov.cn/moh/main).

HEV is the RNA viruses of the about 7.2kb of a kind of nonencapsulated sub-thread normal chain total length, mainly passes through transmission.The icosahedron symmetrical structure spherical virus particle that Bradley etc. are big or small about 27-34nm by immune electron microscopy HEV (Bradley, D.W.et al., Proc Natl Acad Sci U S A, 1987,84 (17): 6277-6281).HEV is containing 3 partly overlapping open reading frames (ORF), 660 amino acid whose polypeptide of ORF2 coding wherein, for viral major structural protein, form viral capsid, key protein while being cell entry cell (Panda SK, Thakral D et al., Rev Med Virol, 2007,17:151).41 Nucleotide in the ORF2 of HEV and ORF1 interval, total length 1980bp, 660 the amino acid whose polypeptide pORF2 that encode, are viral major structural protein, pORF2 can be self-assembled into by the mode of non-disulfide linkage viral capsid (capsid).The translation initial product of ORF2 is the precursor protein (ppORF2) of a 82kD, there is a typical signal peptide sequence in its albumen n end (aa1-aa36), by signal peptide, identify albumen (Signal peptide recognition protein, SRP) enter endoplasmic reticulum (Endoplasmic reticulum, ER), thus in the process of entering, N end signal peptide is sheared and forms ripe pORF2 albumen (72kD).After signal peptide, be one and be rich in arginic region, this strong positive electricity district has been considered to participate in the capsidation (encapisidation) of geneome RNA in virus assembly process.At present existing multiple expression system can be expressed albumen (Jameel, S.et al., J Virol., 1996,70 (1): 207-216 of ORF2 coding; Tyagi, S.et al., Biochem Biophys Res Commun, 2001,284 (3): 614-621; Torresi, J.et al., J Gen Virol., 1999,80 (Pt5): 1185-1188; Panda, S.K.et al., J Clin Microbiol, 1995,33 (10): 2653-2659; Torresi, J.et al., J Virol Methods, 1997,69 (1-2): 81-91; McAtee, C.P.et al., J Chromatogr B Biomed Appl, 1996,685 (1): 91-104; Robinson, R.A.et al., Protein Expr Purif, 1998,12 (1): 75-84; Carl, M.et al., Clin Diagn Lab Immunol, 1994,1 (2): 253-256), the pORF2 albumen of expressing can form not for beta-mercaptoethanol depolymerization but non-covalent homodimer (Zafrullah, M.et al., the J Virol. that can be destroyed by boiling water bath, 1999,73 (5): 4074-4082), show that pORF2 can form homodimer, this homodimer is likely the fundamental unit that forms viral capsid.

The capsid protein gene of HEV is relatively stable, but along with the further variation of encoding gene may cause the variation of viral surface antigen epi-position, thereby cause the difference of serotype, so scientists has been carried out gene type with structural protein coding region (ORF2).The nucleic acid variation that is about to ORF2 district is no more than 20% strain isolated and is classified as a genotype.According to this standard, HEV at least can be divided into 4 main genotype, and wherein 1,3,4 types are Major Epidemic strain, and 2 Xing Zezhi Mexico and African minority area are popular.

For specific detection HEV-3 and HEV-4 pathogenic agent better, find out the specific antibody for HEV-3 and HEV-4, the inventor is in obtaining hepatitis E virus ORF2 on a series of bases with excellent antigenic polypeptide fragment (referring to Chinese patent application CN00130634.0), the homeopeptide fragment of expression and purification HEV-3 and HEV-4, and further obtained the antibody that can secrete specific binding 3 types and 4 type hepatitis E virus ORF2 coded polypeptides and 3 types and 4 type hepatitis E viruss.

Summary of the invention

First aspect present invention relates to antibody or its antigen-binding portion thereof, and they can specific binding 3,4 type hepatitis E viruss and are not combined or weak binding with 1 type hepatitis E virus;

Preferably, the l-asparagine that the epitope of described antibody or the combination of antigen-binding portion thereof institute comprises the 504th, the 490th, 3 type hepatitis E virus ORF2 albumen or 4 type hepatitis E virus ORF2 albumen;

More preferably, the Threonine that the epitope of described antibody institute combination comprises the 489th, 3 type hepatitis E virus ORF2 albumen, the 503rd, 4 type ORF2 albumen, the arginine that 3 the 524th, type ORF2 albumen, 4 type ORF2 albumen are the 538th, and the tyrosine of the 532nd, 3 type ORF2 albumen, the 546th, 4 type ORF2 albumen;

Particularly preferably, the polypeptide fragment that the epitope of described antibody institute combination comprises 3 type hepatitis E virus ORF2 albumen 489th～532 amino acids, or the polypeptide fragment that comprises 4 type hepatitis E virus ORF2 albumen 503rd～546 amino acids.

In embodiments of the invention, described antibody is monoclonal antibody or polyclonal antibody, is preferably monoclonal antibody.

In specific embodiment of the invention scheme, described monoclonal antibody is selected from:

1) by the secreted anti-E type hepatitis virus monoclonal antibody HEV-C3-1 of the hybridoma cell line CCTCC-C201223 that is preserved in Chinese Typical Representative culture collection center;

2) by the secreted anti-E type hepatitis virus monoclonal antibody HEV-C3-2 of the hybridoma cell line CCTCC-C201231 that is preserved in Chinese Typical Representative culture collection center.

Second aspect present invention relates to antibody or its antigen-binding portion thereof, and they can specific binding 3,4 type hepatitis E virus ORF2 albumen, the 394-606 position of the 368-606 position of 3 type ORF2 albumen or 4 types 382-620 amino acids, 3 type ORF2 albumen or 4 type 408-620 amino acids or these protein variant and albumen or its variant corresponding with above-mentioned albumen with 1 type hepatitis E virus is not combined or weak binding;

Preferably, the l-asparagine that the epitope of described antibody or the combination of antigen-binding portion thereof institute comprises the 504th, the 490th, 3 type hepatitis E virus ORF2 albumen or 4 type hepatitis E virus ORF2 albumen, more preferably, the epitope of described antibody institute combination comprises the 489th, 3 type hepatitis E virus ORF2 albumen, the Threonine of 4 the 503rd, type ORF2 albumen, 3 the 524th, type ORF2 albumen, the arginine of 4 the 538th, type ORF2 albumen, with the 532nd, 3 type ORF2 albumen, the tyrosine of 4 the 546th, type ORF2 albumen, particularly preferably, the polypeptide fragment that the epitope of described antibody institute combination comprises 3 type hepatitis E virus ORF2 albumen 489th～532 amino acids, or the polypeptide fragment that comprises 4 type hepatitis E virus ORF2 albumen 503rd～546 amino acids, and/or

Preferably, the K of 368-606 amino acids, the 394-606 amino acids of ORF2 albumen or the variant of these albumen of described antibodies 1 type hepatitis E virus ORF2 albumen, ORF2 albumen _dvalue is higher than the above-mentioned albumen in conjunction with 3,4 type hepatitis E viruss or the K of its variant _dvalue more than 2 times, for example, is 2 times, 4 times, 8 times, more than 16 times; In embodiments of the invention, the K of 368-606 amino acids, the 394-606 amino acid of ORF2 albumen or the variant of these albumen of described antibodies 1 type hepatitis E virus, ORF2 albumen _dvalue is higher than the K of the above-mentioned albumen in conjunction with 3,4 type hepatitis E viruss _dvalue more than 20 times, for example, is 20-100 times; And/or

Preferably, the above-mentioned albumen of described antibodies 3,4 type hepatitis E viruss or the K of its variant _dvalue is less than 1 * 10 ^-8m, for example, be less than 5 * 10 ^-9m.

In embodiments of the invention, the aminoacid sequence of described 3,4 type hepatitis E virus ORF2 albumen is respectively sequence shown in SEQ ID NO:2 or SEQ ID NO:3; The 368-606 amino acids of described 3 type hepatitis E virus ORF2 albumen is sequence shown in SEQ ID NO:5, and the 382-620 amino acids of 4 type hepatitis E virus ORF2 albumen is sequence shown in SEQ ID NO:6; The 394-606 amino acids of described 3 type hepatitis E virus ORF2 albumen is sequence shown in SEQ ID NO:9, and the 408-620 amino acids of 4 type hepatitis E virus ORF2 albumen is sequence shown in SEQ ID NO:8.

In embodiments of the invention, the aminoacid sequence of described 1 type hepatitis E virus ORF2 albumen is sequence shown in SEQ ID NO:1; The 368-606 amino acids of described 1 type hepatitis E virus ORF2 albumen is sequence shown in SEQ ID NO:4; The 394-606 amino acids of described 1 type hepatitis E virus ORF2 albumen is sequence shown in SEQ ID NO:7.

In embodiments of the invention, described 3,4 type hepatitis E virus ORF2 protein variant refer to that the 490th, 3 type ORF2 albumen or 4 types the 504th amino acids are l-asparagine, 3 the 489th, type ORF2 albumen, the 503rd, 4 types are Threonine, 3 the 524th, type ORF2 albumen, the 538th, 4 types are arginine, with the 532nd, 3 type ORF2 albumen, the 546th, 4 types be tyrosine, shown in all the other aminoacid sequences and SEQ ID NO:2 or SEQ ID NO:3, the homology of sequence is more than 85%, for example, more than 90%, for example, more than 95%, for example, more than 99%; The 368-606 position of described 3 type ORF2 albumen, 4 type 382-620 amino acids, the 3 394-606 positions of type ORF2 albumen are, the variant of 4 type 408-620 amino acids refers to that with the correspondence position of ORF2 protein variant be l-asparagine, Threonine, arginine and tyrosine, shown in all the other aminoacid sequences and SEQ ID NO:5,6,8,9, the homology of sequence is more than 85%, for example, more than 90%, for example, more than 95%, for example, more than 99%.

In embodiments of the invention, described 1 type hepatitis E virus ORF2 protein variant refers to that this variant the 490th amino acids is glycine or L-Ala, shown in all the other aminoacid sequences and SEQ ID NO:1, the homology of sequence is more than 85%, for example, more than 90%, for example, more than 95%, for example, more than 99%; The 368-606 amino acids of described ORF2 albumen, the amino acid whose variant of 394-606 of ORF2 albumen refer to that with the correspondence position of ORF2 protein variant be glycine or L-Ala, shown in all the other aminoacid sequences and SEQ ID NO:4,7, the homology of sequence is more than 85%, for example, more than 90%, for example, more than 95%, for example, more than 99%.

Third aspect present invention relates to antibody or its antigen-binding portion thereof, the l-asparagine that the epitope of its combination comprises the 504th, the 490th, 3 type hepatitis E virus ORF2 albumen or 4 type hepatitis E virus ORF2 albumen;

Preferably, the Threonine that the epitope of described antibody institute combination comprises the 489th, 3 type hepatitis E virus ORF2 albumen, the 503rd, 4 type ORF2 albumen, the arginine that 3 the 524th, type ORF2 albumen, 4 type ORF2 albumen are the 538th, and the tyrosine of the 532nd, 3 type ORF2 albumen, the 546th, 4 type ORF2 albumen;

More preferably, the polypeptide fragment that the epitope of described antibody institute combination comprises 3 type hepatitis E virus ORF2 albumen 489th～532 amino acids, or the polypeptide fragment that comprises 4 type hepatitis E virus ORF2 albumen 503rd～546 amino acids.

Fourth aspect present invention relates to hybridoma cell strain, and it is selected from the hybridoma cell strain as next group:

1) be preserved in Chinese Typical Representative culture collection center, the hybridoma cell strain HEV-C3-1 that preserving number is CCTCC-C201223;

2) be preserved in Chinese Typical Representative culture collection center, the hybridoma cell strain HEV-C3-2 that preserving number is CCTCC-C201231.

Fifth aspect present invention relates to antibody or its antigen-binding portion thereof of energy specific binding 3,4 type Hepatitis E Virus Capsid Proteins, described antibody is monoclonal antibody or polyclonal antibody, wherein, described antibody can seal 3,4 described type Hepatitis E Virus Capsid Proteins be selected from that following monoclonal antibody combines active at least 50%, for example at least 60%, at least 70%, at least 80%, for example at least 85%, at least 90%, at least 95%:

1) monoclonal antibody that hybridoma cell strain HEV-C3-1 produces, wherein, hybridoma cell strain HEV-C3-1 is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC-C201223;

2) monoclonal antibody that hybridoma cell strain HEV-C3-2 produces, wherein, hybridoma cell strain HEV-C3-2 is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC-C201231;

Preferably, described antibody be selected from 1) and 2) monoclonal antibody there is identical epi-position.

Sixth aspect present invention relates to the epitope of 3,4 type hepatitis E viruss, and it can and at least comprise the 490th, 3 type hepatitis E virus ORF2 albumen or the l-asparagine of the 504th, 4 type hepatitis E virus ORF2 albumen with the antibody of claim 1-4 or 6 any one or its antigen-binding portion thereof specific binding;

Preferably, the Threonine that described epitope comprises the 489th, 3 type hepatitis E virus ORF2 albumen, the 503rd, 4 type ORF2 albumen, the arginine that 3 the 524th, type ORF2 albumen, 4 type ORF2 albumen are the 538th, and the tyrosine of the 532nd, 3 type ORF2 albumen, the 546th, 4 type ORF2 albumen;

More preferably, the polypeptide fragment that described epitope comprises 3 type hepatitis E virus ORF2 albumen 489th～532 amino acids, or the polypeptide fragment that comprises 4 type hepatitis E virus ORF2 albumen 503rd～546 amino acids.

Seventh aspect present invention relates to composition, and it comprises the present invention first to the antibody of the third aspect or the 5th aspect any one or the epitope of its antigen-binding portion thereof or sixth aspect present invention.

Eighth aspect present invention relates to the method that detects 3,4 type hepatitis E viruss in sample, and it comprises that use the present invention first is to the third aspect or the 5th antibody of aspect any one or the step of its antigen-binding portion thereof.Particularly, said method comprising the steps of:

1) testing sample and the present invention first are contacted to antibody or its antigen-binding portion thereof of the third aspect or the 5th aspect any one;

2) detect described antibody and reacting that sample occurs, preferably, described antibody or its antigen-binding portion thereof also comprise detectable mark.

Preferably, described antibody is monoclonal antibody;

Preferably, described monoclonal antibody is attached in solid phase, and described solid phase is for example selected from as next group: titer plate, magnetic particle, latex particle and nitrocellulose membrane;

Preferably, described monoclonal antibody is to be attached in described solid phase by direction like this, and it can increase the joint efficiency of described monoclonal antibody and described sample.

Preferably, described monoclonal antibody is attached in described solid phase by its constant region.

Preferably, described reaction is measured by enzyme colour developing, fluorescence or chemoluminescence method.

Preferably, described monoclonal antibody is Fab, Fab ', F (ab) 2 or Fv.

Ninth aspect present invention relates to the present invention first to the third aspect or the 5th antibody of aspect any one or the purposes of its antigen-binding portion thereof, it is the existence in sample or its expression level for detection of 3,4 type hepatitis E viruss, or for the preparation of test kit, described test kit is the existence in sample or its expression level for detection of 3,4 type hepatitis E viruss.

Tenth aspect present invention relates to test kit or detection reagent, it comprises the present invention first to the antibody of the third aspect or the 5th aspect any one or the epitope of its antigen-binding portion thereof or sixth aspect present invention, and described test kit or detection reagent are for detection of 3,4 type hepatitis E viruss or diagnosis hepatitis E.

The present invention the tenth relate in one aspect to the present invention first to the antibody of the third aspect or the 5th aspect any one or the epitope of its antigen-binding portion thereof or sixth aspect present invention the purposes in preparing test kit or detection reagent, described test kit or detection reagent for detection of 3,4 type hepatitis E viruss or diagnosis hepatitis E.

The present invention the 12 aspect relates to the present invention first to the antibody of the third aspect or the 5th aspect any one or the epitope of its antigen-binding portion thereof or the sixth aspect present invention purposes in the medicine of preparation prevention or treatment hepatitis E.

The present invention's the tenth three aspects: relates to a kind of method of Dispersal risk, described method comprises utilizes the epitope of 3,4 type hepatitis E viruss or the polypeptide that comprises this epitope or protein to carry out immunity to obtain the step of antibody, and the epitope of described 3,4 type hepatitis E viruss at least comprises the l-asparagine of the 504th, the 490th, 3 type hepatitis E virus ORF2 albumen or 4 type hepatitis E virus ORF2 albumen;

Detailed Description Of The Invention

What unless otherwise defined, all technology used and scientific term all expressed here is the common implication of understanding the those of skill in the art that the present invention relates in field.Here name used and be widely used conventional steps in corresponding field in cell cultures, molecular genetics, accounting chemistry, Immunology Lab operation steps.

" antibody " word in the present invention refers to any one immunoglobulin (Ig), comprise can binding specificity monoclonal antibody, many anti-, dual specifics or the multi-specificity antibody of antigen.A complete antibody comprises two heavy chains and two light chains.Every heavy chain contains a variable region and three constant regions of first, second, third, etc.; Every light chain comprises a variable region and a constant region.Antibody is " Y " type, the second and the 3rd constant region that the neck of " Y " type structure contains two heavy chains, and it forms by disulfide-bonded.Every arm of " Y " type structure contains wherein the first constant region and the variable region of a heavy chain, and the variable region of a light chain and constant region.The variable region of light chain and heavy chain determines the combination of antigen; Three hypervariable regions are all contained in the variable region of every chain, claim that (CDR of light chain (L) comprises LCDR1, LCDR2, LCDR3 in complementary determining region (CDR), the CDR of heavy chain (H) comprises HCDR1, HCDR2, HCDR3(, and it is named by people such as Kabat, see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), 1-3 volume, NIH Publication91-3242, Bethesda Md).Wherein, three CDR are spaced apart by framework region (FR).BiCDR district, framework region is more conservative and form a shelf shape support structure hypervariable region.The constant region of heavy chain and light chain is combined irrelevant with antigen, but has multiple effector function.Antibody can be divided into several classes according to the aminoacid sequence of CH, mainly: IgA, IgD, IgE, IgG and IgM, wherein some class is also further divided into subclass, as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 etc.

Can adopt the hybridoma preparation method of the report in Nature256:495 (1975) such as Kohler to prepare monoclonal antibody.First by immunogen (adding adjuvant in the time of necessity) immunization mouse or other suitable host animal.The injection system of immunogen or adjuvant is generally subcutaneous multi-point injection or abdominal injection.Immunogen in advance lotus root is linked to some known albumen, on serum albumin or soybean pancreatin inhibitor, may contribute to the immunogenicity of enhancement antigen in host.Adjuvant can utilize freund adjuvant or MPL-TDM etc.Animal is subject to after immunity, and the lymphocyte that has the immunogenic antibody of secretion specific binding in body produces.In addition, lymphocyte also can utilize external immunity to obtain.Collect object lymphocyte and myeloma cell and with suitable fusogen, as PEG, merge to obtain hybridoma (Goding, Monoclonal Antibodies:Principles and Practice, pp.59-103, Academic Press, 1996).

The hybridoma of above-mentioned preparation can be inoculated in suitable nutrient solution grows, and preferably contains material that one or more can suppress not merge, parent myeloma cell growth in nutrient solution.For example, to lacking the parent myeloma cell of xanthoglobulin guanine monophosphate transferring enzyme (HGPRT or HPRT), in nutrient solution, add the growth that the materials such as xanthoglobulin, aminopterin-induced syndrome and thymus pyrimidine (HAT substratum) can suppress HGPRT-deficient cells.

It is high that preferred myeloma cell should have fusion rate, and antibody-secreting ability is stable, to abilities such as HAT nutrient solution sensitivities.Wherein, the first-selected mouse of myeloma cell source myelomatosis, strain (THE Salk Institute Cell Distribution Center as derivative in MOP-21 and MC-11 mouse tumor, San Diego, Calif.USA), with SP-2/0 or X63-Ag8-653 cell strain (American Type Culture Collection, Rockville, Md.USA).Also studies have reported that in addition and utilize human myeloma and people mouse allos myeloma cell strain to 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 nutrient solution of Growth of Hybridoma Cell is for detection of the generation of the monoclonal antibody for specific antigen.The binding specificity of measuring the monoclonal antibody of hybridoma generation has immunoprecipitation or external combination test, as radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA).For example, the Scatchard analytical method of utilizing Munson etc. to describe at Anal.Biochem.107:220 (1980) can be used to measure the avidity of monoclonal antibody.

After specificity, avidity and the reactivity of the antibody that hybridoma produces are determined, object cell strain can pass through (Goding, Monoclonal Antibodies:Principles and Practice, pp.59-103, Academic Press, 1996) limiting dilution assay of described standard carries out subcloning.Suitable nutrient solution can be DMEM or RPMI-1640 etc.In addition, the form that hybridoma can also ascitic tumor is grown in animal body.

Utilize traditional immunoglobulin purification method, as albumin A sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography etc., the monoclonal antibody of subclone emiocytosis can be separated from cell culture fluid, ascites or serum.

" antibody " word in the present invention, ultrawhite except refering in particular to complete immune globulin, also the fragment (as being at least an immunocompetence section of immunoglobulin molecules) that refers to immunoglobulin (Ig), as Fab, Fab ', F (ab ') 2, Fv fragment, single-chain antibody molecule or the multi-specificity antibody that formed by any fragment of the immunoglobulin molecules that contains one or more CDR district.In addition, the antibody the present invention relates to antibody that can be also one or more CDR district in a specific human normal immunoglobulin form in conjunction with the framework region of one or more different human normal immunoglobulins.

Monoclonal antibody of the present invention can be the antibody of traditional " Y " type structure shape of comprising two heavy chains and two light chains.In addition, described antibody can be also the Partial Fragment having kept Fab fragment, Fab ', F (ab) 2, Fv or other type on the antibody of traditional " Y " type structure shape of hemagglutinin avidity, and its avidity in conjunction with hemagglutinin can be higher or lower than the antibody of traditional " Y " type structure shape.

Antibody fragment of the present invention can utilize the complete antibody molecule of hydrolysis to obtain (referring to Morimoto et al., J.Biochem.Biophys.Methods24:107-117 (1992) and Brennan et al., Science229:81 (1985)).In addition, these antibody fragments also can directly produce (reviewed in Hudson, Curr.Opin.Immunol.11:548-557 (1999) by recombinant host cell; Little et al., Immunol.Today, 21:364-370 (2000)).Such as, Fab' fragment can directly from E.coli cell, obtain or chemical lotus root connection forms F (ab ') 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).For another example, F (ab ') 2 fragments can connect and obtain with leucine zipper GCN4.In addition, Fv, Fab or F (ab ') 2 fragments also can be directly from recombinant host cell nutrient solution directly separation obtain.Those of ordinary skill in the art knows other technology of Dispersal risk fragment completely.

As used herein, " antigen-binding portion thereof " of term antibody refers to one or more parts of full length antibody, described part keeps the same antigen of binding antibody institute combination, and (for example, ability ERCC1), with the specific binding of complete antibody competition to antigen.Conventionally referring to, Fundamental Immunology, and Ch.7 (Paul, W., ed., the 2nd edition, Raven Press, N.Y. (1989), it is integrated with herein in full by reference with it, for all objects.Can be by recombinant DNA technology or by enzymatic or the chemistry fracture generation antigen-binding portion thereof of complete antibody.In some cases, antigen-binding portion thereof comprises Fab, Fab ', F (ab ') ₂, Fd, Fv, dAb and complementary determining region (CDR) fragment, single-chain antibody (for example, scFv), chimeric antibody, double antibody (diabody) and such polypeptide, at least a portion that it comprises the antibody that is enough to give polypeptid specificity antigen binding capacity.In embodiments of the invention, the Fab fragment that described antigen-binding portion thereof is antibody.

As used herein, term " Fd fragment " means by V _hand C _hthe antibody fragment that 1 structural domain forms; Term " Fv fragment " means the V by the single armed of antibody _land V _hthe antibody fragment that structural domain forms; Term " dAb fragment " means by V _hthe antibody fragment that structural domain forms people such as (, Nature 341:544-546 (1989)) Ward; Term " Fab fragment " means by V _l, V _h, C _land C _hthe antibody fragment that 1 structural domain forms; " Fab ' " fragment refers to the Fab fragment that has comprised part hinge area; Term " F (ab ') ₂fragment " mean to comprise the antibody fragment by two Fab fragments of the disulfide bridge connects on hinge area.

In some cases, the antigen-binding portion thereof of antibody be single-chain antibody (for example, scFv), V wherein _land V _hstructural domain by can be produced as the linker pairing of single polypeptide chain form monovalent molecule (referring to, for example, the people such as Bird, the people such as Science242:423-426 (1988) and Huston, Proc.Natl.Acad.Sci.USA 85:5879-5883 (1988)).This type of scFv molecule can have general structure: NH ₂-V _l-joint-V _h-COOH or NH ₂-V _h-joint-V _l-COOH.Suitable prior art joint is comprised of the GGGGS aminoacid sequence repeating or its variant.For example, can use and there is aminoacid sequence (GGGGS) ₄joint, but also can use its variant (people (1993) such as Holliger, Proc.Natl.Acad.Sci.USA90:6444-6448).Can be used for other joints of the present invention by the people such as Alfthan (1995), Protein Eng.8:725-731, the people such as Choi (2001), Eur.J.Immunol.31:94-106, the people such as Hu (1996), Cancer Res.56:3055-3061, the people such as Kipriyanov (1999), the people such as J.Mol.Biol.293:41-56 and Roovers (2001), Cancer Immunol. describes.

In some cases, antibody is double antibody, that is, and and bivalent antibody, wherein V _hand V _lstructural domain is expressed on single polypeptide chain, but use too short linker so that do not allow to match between two structural domains of same chain, thereby force the complementary structure territory pairing of structural domain and another chain and produce two antigen-binding sites (referring to, for example, the people such as Holliger P., Proc.Natl.Acad.Sci.USA90:6444-6448 (1993), and the people such as Poljak R.J., Structure2:1121-1123 (1994)).

(for example can use routine techniques well known by persons skilled in the art, recombinant DNA technology or enzymatic or chemical break method) for example, from given antibody (monoclonal antibody 2E12), obtain antibody antigen-binding portion thereof (for example, above-mentioned antibody fragment), the antigen-binding portion thereof with regard to specificity screening antibody and in the identical mode of the mode with for complete antibody.

Antibody of the present invention can specific binding 3,4 type hepatitis E viruss.One aspect of the present invention relate to can specific binding 3,4 type Hepatitis E Virus Capsid Proteins and not in conjunction with or the antibody of weak binding 1 type Hepatitis E Virus Capsid Protein.In embodiments of the invention, described antibody is monoclonal antibody.

Anti-3,4 type hepatitis E virus monoclonal antibodies of the present invention are secreted by mouse hybridoma cell strain HEV-C3-1, HEV-C3-2.The title of these monoclonal antibodies is named with its corresponding hybridoma cell strain.Namely, these anti-3,4 type hepatitis E virus monoclonal antibodies are produced by hybridoma cell strain HEV-C3-1, HEV-C3-2 respectively, and difference called after HEV-C3-1, HEV-C3-2.Monoclonal antibody HEV-C3-1, HEV-C3-2 can specific binding 3,4 type Hepatitis E Virus Capsid Proteins.Mouse hybridoma cell strain HEV-C3-1, HEV-C3-2 are at Chinese Typical Representative culture collection center (CCTCC, Wuhan University, Wuhan, China) carry out preservation, preserving number is respectively CCTCC-C201223 (hybridoma cell strain HEV-C3-1, February 20 2012 preservation time), CCTCC-C201231 (hybridoma cell strain HEV-C3-2, February 20 2012 preservation time).

In one embodiment of the invention, relate to a kind of can specific binding as shown in SEQ ID NO:5 hepatitis E virus ORF2 coded polypeptide and not in conjunction with the monoclonal antibody of hepatitis E virus ORF2 coded polypeptide as shown in SEQ ID NO:4.In specific embodiments of the present invention, described monoclonal antibody is the anti-E type hepatitis virus monoclonal antibody HEV-C3-1 that has hybridoma cell line CCTCC-C201223 secreted; Its energy Direct Acquisition 3,4 type HEV virions, but do not catch 1 type HEV virion; The affinity costant of itself and HEV4 type recombinant polypeptide L6E2 is about 1.89 * 10 ^-9, the affinity costant of its Fab fragment is about 6.45 * 10 ^-9, the affinity costant of itself and HEV1 type recombinant polypeptide NE2 is about 4.195 * 10 ^-8, the avidity of its Fab fragment is crossed weak cannot detection.

In yet another embodiment of the invention, described monoclonal antibody is the anti-E type hepatitis virus monoclonal antibody HEV-C3-2 that has hybridoma cell line CCTCC-C201231 secreted; Its energy Direct Acquisition 3,4 type HEV virions, but do not catch 1 type HEV virion; The affinity costant of itself and HEV4 type recombinant polypeptide L6E2 is about 2.984 * 10 ^-10, the affinity costant of its Fab fragment is about 6.69 * 10 ^-9, the affinity costant of itself and HEV1 type recombinant polypeptide NE2 is about 3.75 * 10 ^-8, the avidity of its Fab fragment is crossed weak cannot detection.

The present invention relates to antibody and also comprise and can block monoclonal antibody HEV-C3-1 or HEV-C3-2 in conjunction with the antibody of 3,4 type Hepatitis E Virus Capsid Proteins, for example described antibody is monoclonal antibody.Epi-position on 3,4 type Hepatitis E Virus Capsid Proteins of these monoclonal antibody combinations can be identical with the epi-position that monoclonal antibody HEV-C3-1 or HEV-C3-2 identify.The epi-position that the epi-position that these monoclonal antibodies are identified also can be identified with monoclonal antibody HEV-C3-1 or HEV-C3-2 spatially has overlapping.Such monoclonal antibody can reduce the bonding force at least 50% of monoclonal antibody HEV-C3-1 or HEV-C3-2 and 3,4 type Hepatitis E Virus Capsid Proteins, or at least 60%, or preferably at least 70%, or more preferably at least 75%, or more preferably at least 80%, or more preferably at least 85%, or more preferably at least 90%, or more preferably 95%, or most preferably 99%.

Can adopt ordinary method as Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory, the method of describing in Ed Harlow and David Lane (1988), measures a certain unknown monoclonal antibody and reduces a certain known monoclonal antibody in conjunction with the ability of 3,4 type Hepatitis E Virus Capsid Proteins.For example, first that antigen is pre-coated on microwell plate, then the known monoclonal antibody after the mark of the antibody unlabelled to be measured of serial dilution and certain concentration is jointly added in above-mentioned microwell plate after pre-coated and hatched, after washing, measure known antibodies under different dilution antibody to be measured and be attached to the quantity on plate.The ability of antibody competition known antibodies conjugated antigen to be measured is stronger, and the ability of known antibodies conjugated antigen is just more weak.Conventionally, antigen is pre-coated on 96 hole microwell plates, and utilizes Radio labeled method or enzyme labelling method to measure the unmarked MAbs blocking ability of mark monoclonal antibody.

" antigenic determinant " in the present invention (or claim epi-position) refers in antigen molecule part amino acid or the atomic radical with antibodies.By the chemically reactive surface group of molecule, for example amino acid or carbohydrate or sugared side chain form and conventionally have specific Three Dimensions Structure and a specific charge characteristic conventionally for epi-position or antigenic determinant.For example, epi-position comprises at least 3,4 with unique space conformation conventionally, 5,6,7,8,9,10,11,12, and 13,14 or 15 continuous or discrete amino acid, it can be " linearity " or " conformation ".Referring to, for example, Epitope Mapping Protocols in Methods in Molecular Biology, the 66th volume, G.E.Morris, Ed. (1996).In linear epitope, for example, linear existence of all interactional one-level aminoacid sequence along protein between protein and interacting molecule (antibody).In conformational epitope, interactional point is crossed over the gal4 amino acid residue being separated from each other and is existed, and when interactional point is determined, according to interactional point, determines that the method for conformational epitope is well known in the art.

In embodiments of the invention, the epitope of described 3,4 type hepatitis E viruss is conformation type epi-position.

In specific embodiment of the invention scheme, the epitope of described 3,4 type hepatitis E viruss and the site that antibody HEV-C3-1 and HEV-C3-2 are combined comprise the l-asparagine of the 504th, the 490th, 3 type hepatitis E virus ORF2 albumen or 4 type hepatitis E virus ORF2 albumen, the Threonine of 3 the 489th, type hepatitis E virus ORF2 albumen, the 503rd, 4 type ORF2 albumen, the arginine that 3 the 524th, type ORF2 albumen, 4 type ORF2 albumen are the 538th, and the tyrosine of the 532nd, 3 type ORF2 albumen, the 546th, 4 type ORF2 albumen.

In specific embodiment of the invention scheme, 3 type hepatitis E virus ORF2 albumen 489th～532 amino acids or 4 type hepatitis E virus ORF2 albumen 503rd～546 amino acids, by space folding, form conformation type epi-position.

In specific embodiment of the invention scheme, described epitope can also comprise several amino acid of 3 type hepatitis E virus ORF2 albumen 489th～532 amino acids or 4 type hepatitis E virus ORF2 albumen 503rd～546 amino acids left and right sides, to be conducive to the correct folding of polypeptide, for example, comprise respectively each 1,2,3,4,5 amino acid of the left and right sides.

Can use routine techniques well known by persons skilled in the art, just with the binding competition screening antibodies of identical epi-position.For example, can be at war with and cross competition research, to obtain the antibody of the combination of competition each other or cross competition and antigen.The high throughput method that cross competition based on them obtains in conjunction with the antibody of identical epi-position is described in International Patent Application WO 03/48731.Therefore, can use routine techniques well known by persons skilled in the art, obtain with monoclonal antibody HEV-C3-1 or HEV-C3-2 and compete antibody and the antigen-binding portion thereof thereof in conjunction with the identical epi-position on ORF2 albumen.

As used herein, term " specific binding " refers to the combination of antibody to the epi-position on predetermined antigen.Conventionally, antibody is to be less than about 1 * 10 ^-8m, for example, be less than about 1 * 10 ^-9m, 1 * 10 ^-10m or 1 * 10 ^-11m or less avidity (K _d) conjugated antigen epi-position

As used herein, term " K _d" referring to the combination dissociation equilibrium constant of specific antibodies-AI, it is for describing the binding affinity between antibody and antigen.Equilibrium dissociation constant is less, and antibody-antigen is in conjunction with tightr, and the avidity between antibody and antigen is higher.Conventionally, antibody is to be less than about 1 * 10 ^-8m, for example, be less than about 1 * 10 ^-9m, 1 * 10 ^-10m or 1 * 10 ^-11m or less combination dissociation equilibrium constant (K _d) conjugated antigen, for example, use surface plasma body resonant vibration art (SPR) to measure in BIACORE instrument, or utilize BLI(Bio-Layer Interferometry) technology, follow the tracks of the combination detecting between molecules in solution and biosensor, the variation of the whole process of dissociating, the combination dissociation equilibrium constant of calculating antibody and antigen.

Another aspect of the present invention relates to that a kind of to be present in 3,4 following type hepatitis E virus ORF2 upper and be not present in the antigenic determinant on 1,2 type hepatitis E virus ORF2, described antigenic determinant:

1) can be combined with monoclonal antibody HEV-C3-1; Be exposed to the surface of 3,4 type hepatitis E virus particles; There is conformation dependent, and the dimerization of hepatitis E virus ORF2 polypeptide fragment contributes to the correct formation of this antigenic determinant or fully exposes; Its position of key amino acid that participate in to form this antigenic determinant is the l-asparagine in the 504th, 490 of 3 type hepatitis E virus ORF2 or 4 types, the Threonine of 3 the 489th, type hepatitis E virus ORF2 albumen, the 503rd, 4 type ORF2 albumen, the arginine that 3 the 524th, type ORF2 albumen, 4 type ORF2 albumen are the 538th, with the tyrosine of the 532nd, 3 type ORF2 albumen, the 546th, 4 type ORF2 albumen, or

2) can be combined with monoclonal antibody HEV-C3-2; Be exposed to the surface of 3,4 type hepatitis E virus particles; There is conformation dependent, and the dimerization of hepatitis E virus ORF2 polypeptide fragment contributes to the correct formation of this antigenic determinant or fully exposes; Its position of key amino acid that participate in to form this antigenic determinant is the l-asparagine in the 504th, 490 of 3 type hepatitis E virus ORF2 or 4 types, the Threonine of 3 the 489th, type hepatitis E virus ORF2 albumen, the 503rd, 4 type ORF2 albumen, the arginine that 3 the 524th, type ORF2 albumen, 4 type ORF2 albumen are the 538th, and the tyrosine of the 532nd, 3 type ORF2 albumen, the 546th, 4 type ORF2 albumen.

" sequence same percentage " or " homology " word refer to nucleic acid in candidate sequence or amino acid respectively with corresponding nucleic acid or the per-cent of peptide sequence amplifying nucleic acid or amino acid homogeny.Here the term relevant with nucleotide sequence or peptide sequence " sequence similarity per-cent " relating to be defined as candidate nucleic acid sequence or amino acid residue sequence respectively to the similar per-cent of object nucleotide sequence or aminoacid sequence.For some sequences, are compared in it and aim sequence, can skip sudden change breach if desired, to reach the maximum similar per-cent of gene, and do not go any conservative property of considering similar sequences to suddenly change.The multiple comparison method of this area can be used for determining the similarity of nucleic acid or aminoacid sequence, as available computer software comprises BLAST, and BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) etc.The staff of skilled understands to the suitable measuring parameter of comparison setting, is included as and uses some algorithms of maximum comparability to reach and the comparison of full length sequence.

Those skilled in the art know, can form viruslike particle (for example 1 type hepatitis E virus NE2 polypeptide polymer or 3,4 type hepatitis E virus L6E2 polypeptide polymers, or ORF2 albumen, or in the aminoacid sequence of the polypeptide 368-606 amino acids of ORF2 albumen), can comprise amino acid substitution, interpolation and/or the deletion of one or more conservative types, it still can keep forming character and the immunogenicity of viruslike particle.General, the residue that described polypeptide fragment Semi-polarity amino acid can be all polare Aminosaeren is replaced and is unlikely to change it and forms polymeric ability, and the residue that nonpolar amino acid is all nonpolar amino acid is replaced and is also unlikely to change it and forms polymeric ability.

In the present invention, described antibody or its antigen-binding portion thereof can be with virus in conjunction with referring to that antibody or its antigen-binding portion thereof can catch virus, described not in conjunction with or weak binding refer to and can not catch virus.It is wherein said that to catch viral detection method be well known in the art, for example can be with reference to Narayanan Jothikumar, Theresa L.Cromeans, Betty H.Robertson, X.J.Mengd, Vincent R Hill.A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus.Journal of Virological Methods 131 (2006) 65 – 71.

In the present invention, described antibody or its antigen-binding portion thereof can refer to protein binding the affinity constant (K of antibody or its antigen-binding portion thereof and albumen _d) be less than about 1 * 10 ^-8m, for example, be less than about 1 * 10 ^-9m, 1 * 10 ^-10m or 1 * 10 ^-11m, described not combination or weak binding refer to that antibody or its antigen-binding portion thereof are to be greater than about 1 * 10 ^-8m, for example, be greater than about 1 * 10 ^-7m, 1 * 10 ^-6m or 1 * 10 ^-5m or larger affinity constant (K _d) conjugated antigen epi-position.

In the present invention, described hepatitis E virus ORF2 albumen is identical with the implication of capsid protein, and its aminoacid sequence is for example the sequence shown in sequence SEQ ID NO:1,2,3.

In the present invention, 1, the length of 3 type hepatitis E virus ORF2 albumen is identical, is 660 amino acid, and rear 660 amino acid of 4 type hepatitis E virus ORF2 albumen are corresponding with 1,3 type ORF2 albumen, therefore when amino acid whose position is described, different from 1,3 type hepatitis E viruss.

In the present invention; 368-606 amino acids, the 4 type 382-620 amino acids of described 1 type, 3 type hepatitis E virus ORF2 albumen can form viruslike particle in expression and purification process; and have that good neutralizing epitope is active, immunogenicity and immune protective; by transmission electron microscope and atomic force microscope, it is analyzed; find that this viruslike particle is the regular spherical particle of diameter 13.5-23.0nm, its concrete molecular weight is indefinite.

In the present invention, the 394-606 amino acids of described 1 type, 3 type hepatitis E virus ORF2 albumen, 4 type 408-620 amino acids can form in expression and purification process take the multiple polymer form that dimer is basic structure, NE2 and L6E2 antigen well reproduced the main space constitutional features on HEV viral capsid surface, comprise at least 2 neutralizing sites and immunodominant epitope (referring to Li Shaowei. the research in Hepatitis E Virus Capsid Protein assembled in vitro dependency structure territory. the doctor of Xiamen University Diplomarbeit, 2003).

In the present invention, the K between described antibody and antigen _dvalue is measured by Octet RED96 protein interaction workstation, it utilizes BLI(Bio-Layer Interferometry) technology, can follow the tracks of the combination detecting between molecules in solution and biosensor, the variation of the whole process of dissociating, the combination dissociation equilibrium constant of calculating antibody and antigen.

The beneficial effect of the invention

The invention provides the antibody of can specific binding 3,4 type hepatitis E viruss and not being combined with 1 type hepatitis E virus, and with the epitope of described antibodies.Utilize antibody of the present invention and epitope can detect the existence of 3,4 type hepatitis E viruss or its antibody, can be used for diagnosis and the treatment of hepatitis E.

Preservation statement

Mouse hybridoma cell strain HEV-C3-1, HEV-C3-2 are at Chinese Typical Representative culture collection center (CCTCC, Wuhan University, Wuhan, China) carry out preservation, preserving number is respectively CCTCC-C201223 (hybridoma cell strain HEV-C3-1, February 20 2012 preservation time), CCTCC-C201231 (hybridoma cell strain HEV-C3-2, February 20 2012 preservation time).

Accompanying drawing explanation

The combination of Fig. 1 monoclonal antibody and different shaped hepatitis E virus detects

The Western blot that Fig. 2 antibodies epi-position conformation is analyzed detects

Embodiment

Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.In the present invention, all to take the ORF2 albumen of hepatitis E virus be benchmark to amino acid whose Position Number.

Embodiment 1: the preparation of anti-HEV capsid protein p239 monoclonal antibody

The preparation of different shaped HEV capsid protein antigen:

HEV is divided into 4 main genotype, and wherein gene 1,3,4 types are Major Epidemic strain, and 2 XingHEV Mexico were broken out once, and has the representative strain of a strain, only has in addition a small amount of discovery.Respectively at transferring the expressing gene of transferring ORF2a.a.382-620 section in ORF2a.a.368-606 section, 4 C-type virus Cs in HEV1,3 C-type virus Cs, be structured on expression vector, utilize escherichia expression system, the p239 albumen of having prepared 1,3,4 type HEV sources, difference called after p239 (1), p239 (3), p239 (4), its aminoacid sequence is respectively as shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.

Small white mouse: 6 week age, company of female Balb/c Shu Wei Shanghai Slac Experimental Animal Co., Ltd. bought.

The preparation of hybridoma:

In the body of use standard, immunization ways and PEG fusion method obtain monoclonal antibody, detailed method is referring to Ed Harlow et al., " Antibodies A Laboratory Manual ", the concise and to the point process of Cold Spring Harbor Laboratory 1988. is as follows:

Mouse immune: by above-mentioned p239 (3), p239 (4) and Fu Shi Freund's complete adjuvant (CFA) equal-volume mixing and emulsifying, through the subcutaneous multi-point injection of four limbs and the head back of the body, every per injection 1mg/ml albumen 300ul.15d and 29d after first immunisation, use respectively the HEV capsid protein antigen p239 (3) of same dosage and p239 (4) to add Freund's incomplete adjuvant (IFA) and carry out booster immunization.After booster immunization, for blood sampling, indirect elisa method detects the reaction titre of Serum Antibody and p239 (3) and p239 (4) for the second time, when titre reaches after 1:100000, gets mouse spleen and does and merge.Merge front 72hr booster immunization again.Prepare 10 and merge plate.

Merge: get Serum Antibody and merge mutually with murine myeloma cell SP2/0 with the highest mouse spleen cell of the reaction titre of p239 (4) with p239 (3).First spleen is ground and obtains splenocyte suspension, then with the mixing in the SP2/0 of logarithmic phase murine myeloma cell of low ten times of cell count, through PEG1500 effect 1min by two kinds of cytogamy together, then fused cell liquid 100ml is divided to install in 10 96 orifice plates and cultivate.Merge substratum for the complete screening culture medium of RPMI1640 containing HAT and 20%FBS.Antigen-specific sex clone is screened by indirect elisa method, and screening has strong reactivity with p239 (3) and p239 (4), and with the cell strain of monoclonal antibody of p239 (1) weak reactivity.After 3 time clonings, obtain stable cell strain of monoclonal antibody.

The screening of hybridoma: after merging, cell is cultivated after 10 days on 96 porocyte plates, absorption cell conditioned medium is done with the titre of reacting of HEV capsid protein antigen and is detected, screening has strong reactivity with p239 (3) and p239 (4), and with the weakly reactive cell strain of monoclonal antibody of p239 (1).Cloning is continued in positive hole, until the secreted antibody of cell strain can stably specially react with HEV capsid protein p239 (3) and p239 (4).Screen two strain of hybridoma strain HEV-C3-1, HEV-C3-2.

The selection result: obtain two strain specific reaction monoclonal antibody HEV-C3-1, HEV-C3-2.

The cultivation of hybridoma: stable authentic monoclonal antibody cell strain is amplification cultivation in CO2gas incubator first, is transferred to 24 holes through 96 holes, transfers to 50ml cell bottle through amplification cultivation.Then the cell in collecting cell bottle is expelled in mouse peritoneal, draws ascites after 7-10 days from mouse peritoneal.

The purifying of monoclonal antibody:

Ascites is first processed with 50% ammonium sulfate precipitation, and then to PB, pH7.4 dialysis, uses DEAE post purifying under HPLC afterwards, obtains the monoclonal antibody after purifying, the monoclonal antibody purity after SDS-PAGE purification Identification.

Monoclonal antibody and the checking of different shaped HEV capsid protein specific reaction:

Adopt indirect ELISA method to verify, coated recombinant antigen p239 (1), p239 (3), , the every hole 100ng of p239 (4), antibody dilution to 1 μ g/ml to be checked is added to 100 μ L as original titre, and 10 times of gradient dilutions, with the sheep anti-mouse igg 1:5000 dilution of horseradish peroxidase-labeled, add 100 μ L anti-as detecting two, the ELISA value of reading is greater than 0.1 for detecting the positive, analyze the titre of reacting of antibody and different shaped antigen, relatively specific antibody respectively with different shaped HEV capsid protein specific reaction, result is as table 1, list HEV-C3-1, HEV-C3-2 and p239 (1), p239 (3), the reaction titre of p239 (4), visible two strain monoclonal antibodies all have good specificity, or weak react reactionless with p239 (1), and stronger to the reactivity of p239 (3) and p239 (4), while being 0.01ng to antigen add-on, still can detect.

Table 1 monoclonal antibody and different shaped HEV capsid protein atopy

Wherein antibody 8G12,15B2 are two strain control antibodies, for the mouse source antibody for p239, reactive consistent with different shaped p239, referring to Shuizhen He, Ji Miao, Zizheng Zheng, et al.Putative receptor-binding sites of hepatitis E virus.Journal of General Virology (2008), 89,245 – 249.

The capture ability of monoclonal antibody to different shaped hepatitis E virus:

Use 20mM PB, the concentration that pH7.4 is diluted to 3 μ g/100 μ L monoclonal antibody is coated in the micropore of polystyrene board, the 300 every holes of μ L, and 4 ℃ are coated with 10 hours, and then 37 ℃ are coated with 1 hour, and PBST washes 1 time.Add containing 2%(mass/volume ratio) the PBS350 μ L sealing of BSA, hatch 2 hours for 37 ℃.Then add virus quantity to be all 1 * 10 ⁵the 200 μ L1 of PFU, 3,4 type hepatitis E virus suspensions, hatch 2 hours for 37 °.The plate of having hatched is washed plate 5 times.Wash after plate, every hole adds 200 μ LTrizol cracking, after 4 ℃ of cracking 10min, extract the RNA of the hepatitis E virus in sample, carry out Real-timePCR quantitative experiment, result is as Fig. 1, and the combination of visible HEV-C3-1 and this two strains monoclonal antibody of HEV-C3-2 and hepatitis E virus has very strong type specificity.Same viral add-on, and 1 type HEV of monoclonal antibody combination only has 200 copies even lower than real-timePCR detection level lower limit, and and 3 types and the 4 type HEV of monoclonal antibody combination basically identical, in being about the level of 10000 copies.

The affinity constant of monoclonal antibody and Fab thereof and different shaped antigen detects:

Monoclonal antibody Fab enzyme is cut: the monoclonal antibody of purifying 20mmol/L PB(pH7.0) damping fluid dialysed overnight, the papoid of 10mg/mL is diluted to 1mg/mL with same buffer, and include 10mmol/L EDTA, the enzyme liquid of 10mmol/L halfcystine.Enzyme liquid is added in the antibody after dialysis, with regard to the quality of enzyme and antibody when the combination condition of 37 ℃ of water-bath times carry out a small amount of and grope, obtain abzyme to be cut completely after mass ratio and time conditions, carry out great many of experiments, after water-bath completes, add the standing 1h of iodo-acid amide (final concentration 30mmol/L) lucifuge, rear termination endonuclease reaction.

The purifying of monoclonal antibody Fab fragment: the monoclonal antibody that enzyme is cut is to 20mmol/LTris-HCl(pH8.0) damping fluid dialysed overnight, then utilizes DEAE-HPLC purifying.20mmol/LTris-HCl(pH8.0) balance pillar loading, continue to wash post with low salt buffer, until UV280 absorption value changes, is less than 0.1mAU, collects and penetrate peak, is the Fab fragment of purifying.Applied sample amount 10-30mg antibody, flow velocity 1ml/min, detects wavelength 280nm.Degerming (0.22 μ m millipore filtration) packing after the ultrafiltration and concentration pipe for Fab (Millipore) collected is concentrated, be stored in 4 ℃ standby.

Affinity constant detects: by upper 394 to 606 213 aminoacid sequences of NE2(1 type HEV ORF2, SEQ ID NO:7) and upper 408 to 620 213 aminoacid sequences of L6E2(4 type HEV ORF2, SEQ ID NO:8) (NE2 polypeptide and L6E2 polypeptide are ORF2 a.a.394-606 to albumen, 408-620 section amino acid, in escherichia expression system, can form polymer form, and well reproduced the main space structure on HEV viral capsid surface, upper 394 to 606 213 aminoacid sequences of 3 type HEV ORF2 are as shown in SEQ ID NO:9) be diluted to 100ug/mL, be 1:5(antigen in molar ratio: ratio vitamin H) adds corresponding vitamin H (Biotin), the standing 1h of normal temperature after mixing, after dialyse to PBS damping fluid, in-20 ℃, save backup.Use Octet Red 96 systems (U.S. Fortebio company) antigen and monoclonal antibody to be carried out to the dynamic analysis (carrying out according to operational manual) of combination, at rotating speed, be all to carry out under 1000rpm, the damping fluid environment that is PBS in steps.Balance 10min in PBS is standby for probe, and the biotin labeled antigen that is 25 μ g/mL by concentration is fixed on Streptavidin (SA) Biosensor(U.S. Fortebio company) on.Program is as follows: baseline (baseline) 120s, be written into (loading) 300s, and baseline (baseline2) 120s, in conjunction with (association) 600s, (dissociation) 900s dissociates.Data acquisition and analysis software is respectively Data Acquisition v6.4 and Data Analysis v6.4.Adopt the pattern of 1:1 to carry out relative binding kinetics analysis.The avidity result of HEV-C3-1, HEV-C3-2 and NE2, L6E2 is as table 2, and visible two strain monoclonal antibodies all have good specificity, are all greater than 10 with NE2 avidity ^-8m; And stronger to the avidity of L6E2, K _dvalue is all less than 5 * 10 ^-9m.

Table 2 monoclonal antibody and different shaped HEV capsid protein affinity constant

"-" represents to be greater than system upper limit of detection lower than avidity numerical value, and (upper limit of detection is 10 ^-3m)

Embodiment 2: monoclonal antibody and 3,4 type hepatitis E viruss are combined epitope analysis

In different shaped hepatitis E virus ORF2, p239 protein type conservative amino acid is analyzed:

Utilize ncbi database by the 159 strain complete genome sequences of having reported, cut the gene of p239 albumen in the ORF2 that to encode, minute 1,2,3,4 types are compared, and find out in its type and guard unanimously, and 1, the 2 types amino acid different from 3,4 types.As table 3,490 amino acids wherein, in 1 C-type virus C, finding that there is 1 strain virus amino acid is L-Ala (A), all the other 23 strain virus are glycine (G), because L-Ala and glycine belong to aliphatic category amino acid together, differ a methyl, difference is little, is classified as conservative amino acid.The binding ability of host's difference of different shaped hepatitis E virus and HEV-C3-1, HEV-C3-2 monoclonal antibody and 3,4 type hepatitis E viruss may be caused by these five special conservative amino acid of type by force.

In table 3 different shaped hepatitis E virus ORF2, p239 protein type conservative amino acid is analyzed

The design preparation of mutain:

Utilize the method for rite-directed mutagenesis, on the basis of p239 (4) by 504 single-points the glycine mutation to p239 (1), the mutain obtaining is labeled as p239 (4)-504.P239 (4)-504 sports glycine (G) by l-asparagine (N) for the 404th amino acids corresponding to 4 type hepatitis E virus ORF2 albumen of p239 (4) (being the 123rd amino acids of p239), and this position is corresponding to the 490th amino acids of 1,3 type hepatitis E virus ORF2 albumen.

The Responsiveness of monoclonal antibody and mutain

3 kinds of genotypic wild-type p239, mutain p239 (4)-504 with the reactive detection method of HEV-C3-1, HEV-C3-2 and 2 strain control antibodies 8G12,15B2 referring to embodiment 1, reactivity mark between monoclonal antibody and p239 (4) turns to 1, is converted into relative response value with the reactivity of p239 (1), p239 (3) and other mutains.Relative response value is got and be take 2 powers that are the end, and the numerical value n obtaining represents that corresponding antibody-antigen reactivity is this strain antibody and p239 (4) reactive 2 ⁿdoubly.N value the results are shown in Table 4, visible on wild-type protein HEV-C3-1 compare with control antibodies and just have very strong otherness with the reactivity of HEV-C3-2, with the reactivity of p239 (3) and p239 (4) reactivity far above p239 (1), and control antibodies 8G12 and 15B2 do not have difference substantially to 3 kinds of genotypic wild-type p239 reactivities.On the basis of p239 (4), the simple point mutation albumen of 5 points (is on the basis of p239 (4) to the amino acid mutation to p239 (1), referring to table 3) can find out that with the reactivity of HEV-C3-1, HEV-C3-2 what this epitope antibodies was had the greatest impact is 490,3 type, 4 type 504 amino acids, the l-asparagine that can determine 490,3 type, 504,4 type be on 3,4 type Hepatitis E Virus Capsid Proteins with the key amino acid of the special antigenic determinant of 1 type Hepatitis E Virus Capsid Protein.

The reactivity of table 4 monoclonal antibody and mutain (n value)

Embodiment 3 specific antibodies are analyzed in conjunction with the conformation of epi-position

The L6E210 μ l boiling is splined on to 10%SDS-polyacrylamide gel electrophoresis and carries out electrophoresis, then 35mA electric current carries out transferring film 1h.After transferring film, add 4 ℃ of sealings of 5% skimmed milk to spend the night.TNT washes film 3 times, each 10min.The antibody to be checked that again 1:2000 is diluted to 1XTN adds in film, and under room temperature, shaking table is hatched 1h.TNT washes film 3 times, each 10min.With the sheep anti-mouse antibody 1:5000 dilution of horseradish peroxidase-labeled, add conduct detection two anti-, under room temperature, shaking table is hatched 1h.TNT washes film 3 times, each 10min.Colour developing, takes pictures.Result is as Fig. 2, and for by boiling the L6E2 albumen that destroys conformation, HEV-C3-1, HEV-C3-2 and 8G12 antibody lose reactivity, and 15B2 still exists reactivity.Illustrate HEV-C3-1, HEV-C3-2 and 8G12 antibody for epi-position be conformational epitope, and 15B2 for be linear epitope.

Embodiment 4 specific antibodies are in conjunction with the related amino acid analysis of epi-position

Choose the amino acid sites that is positioned at 4 type Hepatitis E Virus Capsid Protein surfaces, these amino acid sites are independently sported respectively to L-Ala (A), prepare these mutains, by the method consistent with " Responsiveness of monoclonal antibody and mutain " in embodiment 2, test, experimental result is as table 5, visible specific antibody HEV-C3-1 and HEV-C3-2 are responsive to the sudden change of 4 type hepatitis E virus ORF2 albumen the 538th, 503,546 amino acids, illustrate that the epi-position of specific antibody combination also comprises the 538th, 503,546 amino acids.The epitope that antibody HEV-C3-1 is combined with HEV-C3-2 be can release thus and 3 type hepatitis E virus ORF2 albumen 489th～532 amino acids, 4 type hepatitis E virus ORF2 albumen 503rd～546 amino acids comprised.

Table 5 specific antibody is in conjunction with the related amino acid analysis of epi-position

Embodiment 5 monoclonal antibodies are tested for the rhesus monkey of specific detection 3,4 type hepatitis e virus infections:

Get and infect respectively 1,4 type hepatitis E viruss and the rhesus monkey ight soil sample 1g in the toxin expelling phase, resuspended with 5mL PBS, after the centrifugal 5min of 12000rpm, get supernatant, make ight soil and treat sample.

Adopt sandwich ELISA method to verify, the every hole 800ng of Sheet clonal antibody HEV-C3-1 and HEV-C3-2, adds in the rhesus monkey ight soil sample of toxin expelling phase and treats sample 100 μ L, and real-timePCR detects in 100 μ L ight soil samples and is 10 containing virus quantity ⁷copy number, anti-(for the 4# antibody of the mark horseradish peroxidase of hepatitis E virus antibody with the reaction two in hepatitis E virus antigen detection kit (Beijing Tso Biological Pharmaceutical Co), HEV-0396) anti-as detecting two, analyze the reactivity of antibody and different shaped antigen, compare the detectivity of specific antibody to different shaped hepatitis E virus, result is as table 6, visible this two strain antibody can specificly detect the hepatitis E virus of 4 types, thereby can be used for the diagnosis to 4 type hepatitis E patients.

The detectivity of table 6 monoclonal antibody to 1,4 C-type virus Cs

Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.

Claims

1. antibody or its antigen-binding portion thereof, they can specific binding 3,4 type hepatitis E viruss and are not combined or weak binding with 1 type hepatitis E virus;

Preferably, the l-asparagine that the epitope of described antibody institute combination comprises the 504th, the 490th, 3 type hepatitis E virus ORF2 albumen or 4 type hepatitis E virus ORF2 albumen;

2. antibody or its antigen-binding portion thereof, they can specific binding 3,4 type hepatitis E virus ORF2 albumen, 368-606 position or 4 type 382-620 amino acids, the 3 394-606 positions of type ORF2 albumen or the variant of 4 type 408-620 amino acids or these albumen of 3 type ORF2 albumen, and albumen or its variant corresponding with above-mentioned albumen with 1 type hepatitis E virus are not combined or weak binding;

Preferably, the l-asparagine that the epitope of described antibody institute combination comprises the 504th, the 490th, 3 type hepatitis E virus ORF2 albumen or 4 type hepatitis E virus ORF2 albumen, more preferably, the epitope of described antibody institute combination comprises the 489th, 3 type hepatitis E virus ORF2 albumen, the Threonine of 4 the 503rd, type ORF2 albumen, 3 the 524th, type ORF2 albumen, the arginine of 4 the 538th, type ORF2 albumen, with the 532nd, 3 type ORF2 albumen, the tyrosine of 4 the 546th, type ORF2 albumen, particularly preferably, the polypeptide fragment that the epitope of described antibody institute combination comprises 3 type hepatitis E virus ORF2 albumen 489th～532 amino acids, or the polypeptide fragment that comprises 4 type hepatitis E virus ORF2 albumen 503rd～546 amino acids, and/or

Preferably, the K of 368-606 amino acids, the 394-606 amino acids of ORF2 albumen or the variant of these albumen of described antibodies 1 type hepatitis E virus ORF2 albumen, ORF2 albumen _dvalue is higher than the above-mentioned albumen in conjunction with 3,4 type hepatitis E viruss or the K of its variant _dvalue more than 2 times, for example, is 2 times, 4 times, 8 times, more than 16 times; And/or

Preferably, the K of the above-mentioned albumen of described antibodies 3,4 type hepatitis E viruss or the variant of these albumen _dvalue is less than 1 * 10 ^-8m, for example, be less than 5 * 10 ^-9m.

3. antibody or its antigen-binding portion thereof, the l-asparagine that the epitope of its combination comprises the 504th, the 490th, 3 type hepatitis E virus ORF2 albumen or 4 type hepatitis E virus ORF2 albumen;

4. the antibody of claim 1-3 any one or its antigen-binding portion thereof, wherein said antibody is selected from:

5. hybridoma cell strain, it is selected from the hybridoma cell strain as next group:

6. can specific binding antibody or its antigen-binding portion thereof of 3,4 type Hepatitis E Virus Capsid Proteins, described antibody is monoclonal antibody or polyclonal antibody, wherein, described antibody can seal 3,4 described type Hepatitis E Virus Capsid Proteins be selected from that following monoclonal antibody combines active at least 50%, for example at least 60%, at least 70%, at least 80%, for example at least 85%, at least 90%, at least 95%:

7.3, the epitope of 4 type hepatitis E viruss, its can with antibody or its antigen-binding portion thereof specific binding of claim 1-4 or 6 any one, and at least comprise the l-asparagine of the 504th, the 490th, 3 type hepatitis E virus ORF2 albumen or 4 type hepatitis E virus ORF2 albumen;

8. composition, the antibody that it comprises claim 1-4 or 6 any one or the epitope of its antigen-binding portion thereof or claim 7.

9. the method for 3,4 type hepatitis E viruss in detection sample, it comprises that right to use requires 1-4 or the antibody of 6 any one or the step of its antigen-binding portion thereof.

10. claim 1-4 or the antibody of 6 any one or the purposes of its antigen-binding portion thereof, it is the existence in sample or its expression level for detection of 3,4 type hepatitis E viruss, or for the preparation of test kit, described test kit is the existence in sample or its expression level for detection of 3,4 type hepatitis E viruss.

11. test kits or detection reagent, the antibody that it comprises claim 1-4 or 6 any one or the epitope of its antigen-binding portion thereof or claim 7, described test kit or detection reagent are for detection of 3,4 type hepatitis E viruss or diagnosis hepatitis E.

The antibody of 12. claim 1-4 or 6 any one or the purposes of the epitope of its antigen-binding portion thereof or claim 7 in preparing test kit or detection reagent, described test kit or detection reagent are for detection of 3,4 type hepatitis E viruss or diagnosis hepatitis E.

13. the purposes of the epitope of the antibody of claim 1-4 or 6 any one or its antigen-binding portion thereof or claim 7 in the medicine of preparation prevention or treatment hepatitis E.

The method of 14. 1 kinds of Dispersal risks, described method comprises utilizes the epitope of 3,4 type hepatitis E viruss or the polypeptide that comprises this epitope or protein to carry out immunity to obtain the step of antibody, and the epitope of described 3,4 type hepatitis E viruss at least comprises the l-asparagine of the 504th, the 490th, 3 type hepatitis E virus ORF2 albumen or 4 type hepatitis E virus ORF2 albumen;