CN101492495A - A group of antigen epitope polypeptide and uses thereof - Google Patents

Abstract

The invention discloses a set of epitope polypeptides, particularly relates to a neutralizing B cell epitope sequence of encephalitis B virus E protein, and further discloses application of controlling and diagnosing encephalitis B virus by these epitopes. The amino acid sequences of the epitope polypeptides in the invention are respectively any amino acid sequence of SEQ ID NO: 64, SEQ ID NO: 79, SEQ ID NO: 95, SEQ ID NO: 108, SEQ ID NO: 124 and SEQ ID NO: 139. After the epitope polypeptides in the invention are coupled or fused with carrier proteins to be expressed as immunogenic or vaccine immuno animal organism, neutralizing antibodies aiming at JEV can be generated, and the JEV can be neutralized in vivo or in vitro so as to prevent virus from infecting animal organism. The epitope polypeptides in the invention or the junctional complex thereof can be used as reagents for detecting encephalitis B virus antibodies or encephalitis B virus polypeptide antibodies.

Description

One group of antigen epitope polypeptide and application thereof

Technical field

The present invention relates to antigen epitope polypeptide, relate in particular to encephalitis b virus E albumen neutrality B cell antigen epitope polypeptide, the invention still further relates to the application of this antigen epitope polypeptide in control and diagnosis encephalitis b virus, belong to the molecular immunology field.

Background technology

The epidemic encephalitis type B encephalitis B is that (Japanese Encephalitis virus, a kind of important mosquito matchmaker property people beast that JEV) causes suffers from transmissible disease altogether by encephalitis b virus.This virus is at first separated acquisition in nineteen fifty-three in Japan from patient's cerebral tissue, therefore claim japanese encephalitis virus, and associated diseases claims Japanese B encephalitis in Japan.Separated this virus in China,, name and be epidemic encephalitis type B, be called for short encephalitis in order to distinguish mutually with encephalitis A early than 1940.Worldwide have 30 every year, 000-50,000 routine encephalitis case, about 25%-30% death, 50% causes permanent central nervous system sequela.The annual in recent years encephalitis case of China is greater than 5000 examples, more than dead 200 examples.For the multiple animal susceptible of epidemic encephalitis B virus, but except that people, horse and pig, do not present clinical symptom usually.The people is mainly caused the acute infection of central nervous system, and it is feature with high heat, the disturbance of consciousness, tic, respiratory insufficiency and meningeal irritation sign clinically that the people infects.Sow infects can cause miscarriage, produce stillborn foetus or mummy tire, and it is feature that the boar infection causes the testis enlargement to influence reproductive performance, and the minority pig only infects and may present heating and nervous symptoms, and other pigs then great majority do not manifest symptom.Cause enormous economic loss to pig industry.Equus easy infection, minority cause encephalitis and nervous symptoms, and horse is the final host.Mosquito is the main communication media of JEV, and pig is that this virus is most important storage of occurring in nature and propagation host.Encephalitis b virus is mainly popular in the Asia, and recent findings also has report in the Southern Hemisphere, and this explanation epidemic encephalitis type B may worldwide threaten human health.

Encephalitis b virus belongs to flaviviridae, and Flavivirus is the strand positive chain RNA virus.The about 11kb of genome comprises a big open reading frame (ORF), and two ends are 5 ' end and 3 ' end non-coding region (UTR).Genome 5 ' end has cap sequence, but does not have poly (A) tail at 3 ' end.ORF coding contains 3432 amino acid whose precursor proteins, is processed as 10 maturation proteins under the effect of virus and host cell proteins enzyme, comprise 3 structural protein (C, prM, E), 7 Nonstructural Proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).Virus particle is spherical in shape, and the about 30nm of diameter has cyst membrane.JEV has only a serotype, belongs to same serogroups with western sieve virus, black Luo Hegu encephalitis, St. Louis encephalitis virus etc. on antigenicity, has antigenic cross-reaction.Virus envelope glycoprotein has the blood clotting characteristic, animal erythrocytes such as energy aggegation goose, dove, chick.Virus is to a little less than the heat resist power, and 56 ℃ of 30 minutes or 100 ℃ got final product deactivation in 2 minutes, but very strong to low temperature and exsiccant resistibility.Ether, 1: but the equal inactivation of viruses of 1000 sodium deoxycholate and common disinfectants.Unstable under acidic condition, appropriate pH 8.5～9.0.

Adopting vaccine to carry out preventive vaccination is these disease popular effective measure of control.To the research of encephalitis b virus epitope is exploitation effective gene engineered vaccine and the research theoretical basis based on the diagnostic detect reagent of epitope.E albumen is a kind of important structural protein of encephalitis b virus particle surface, is important immune protective antigen.The B cell antigen epi-position that has neutralizing effect in protective antigen plays an important role in stimulating body generation neutrality antibody.Identify that correctly and at length artificial vaccine that epitope has no side effect to the diagnosis of disease, design and immunotherapeutic agent etc. have important meaning.

Reported to some extent for the research of encephalitis b virus E proteantigen epi-position in the past, (KOLASKAR A S such as Kolaskar, KULKARNI-KALE U.Prediction of Three-DimensionalStructure and Mapping of Conformational Epitopes of Envelope Glycoprotein ofJapanese Encephalitis Virus.Virology, 1999,261 (1): 31～42) by three-dimensional structure prediction to E proteantigen epi-position, think that the proteic three-dimensional structure of E mainly is made up of 3 structural domains, structural domain I has serology and bioactive epitope; Domain II has the active and hemagglutination activity epitope of neutralization; Wherein domain II I is folded into the immunoglobulin (Ig) spline structure at virus surface independently, because this conformation, many antigen neutralizing epitopes all concentrate on this structural domain; Kolaskar and Urmila (Kolaskar c b A S, Urmila K K.Prediction of Three-Dimensional structure and mapping ofconformational epitopes of envelope glycoprotein of Japanese encephalitis virus.Virology, 1999,261:31～42.) with monoclonal antibody JEV E albumen is carried out antigen site analysis revealed E albumen and have 6 antigenic determinants at least: flaviviridae specific antigens decision family, can induce to produce HI antibody, not produce neutralizing antibody (NA); Subgroup specific antigens determinant has only NA, and no HI produces; Subgroup specific antigens determinant has HI and NA simultaneously; Subgroup specific antigens determinant, no HI and NA, but energy and JEV, West Nile virus etc. react; The species-specific antigen determinant has only NA, can only react with JEV.But be limited to research level at that time, these researchs all do not have the research of concrete epitope sequence information.The analysis that other research about the encephalitis B epitope focuses mostly on and carries out in the monoclonal antibody of conformation type epi-position, as (Kimura-Kuroda J such as Kimura-Kmoda, Yasui K.Topographical analysis of antigenic determinants on envelope glycoprotein V3 (E) of Japanese encephalitis virus, using monoclonal antibodies.J Virol.1983Jan; 45 (1): 124-32.) use 10 strain monoclonal antibodies JEV E proteantigen determinant is analyzed, the result shows have 5 groups of different antigenic determinants at least on E albumen, promptly the 1st group, flavivirus HI (hemagglutination-inhibition test, hemagglutination inhibition) cross reaction, no neutralizing antibody; The 2nd group, subgroup HI specific reaction, no neutralizing antibody; The 3rd group, low HI has neutralizing antibody; The 4th group and 5 groups is not had HI, neutralizing antibody is arranged.The 1st group site and other group obvious difference wherein, the 2nd and 3 group site is approaching.This result also show the HI site with in and the site be what to separate; And have 2 different HI sites, and one is the flavivirus cross reaction, another is that subgroup is special.Though these researchs confirm to have the conformation epitope on JEV and other flavivirus E albumen with the monoclonal antibody CBA, fail to determine its amino acid composition.Because conformational epitope is through certain folding formed structure that has biological function on space conformation by discontinuous amino-acid residue in the protein, structural research to conformational epitope at present is very difficult, is difficult to certain conformational epitope of research is which amino-acid residue in the protein primary sequence to be made of.Utilization peptide storehouse triage techniques can screen the mimic epitopes of conformational epitope, (Lin C W such as Lin, Wu S C.Identification of mimotopes of the Japanese encephalitis virus envelope proteinusing phage-displayed combinatorial peptide library.J Mol Microbiol Biotechnol, 2004,8 (1): 34～42.) use the mimic epitopes that phage display peptide library has been identified the JEV envelope protein, have (D/E) (Y/T/S) X (2) of a conservative motif X (1) with monoclonal antibody 2H2 bonded mimic epitopes, this motif is positioned at the outer surface of E albumen zone III, but the mimic epitopes of gained can only be approaching with true epi-position on function, and can not analyze the real structure information of conformational epitope in the antigen.

Linear epitope in the epitope is made up of successive amino-acid residue in the proteantigen, and this epi-position still can be by antibody recognition in metaprotein.Because linear epitope, the concrete sequential structure information of Analysis and Identification linear epitope is more or less freely.Linear epitope also is to be easier to be directly used in related application researchs such as diagnosis, recombinant vaccine, synthetic peptide vaccine simultaneously.Also report to some extent for encephalitis b virus E albumen linear epitope, (Seif S A such as Seif, Morita K, Matsuo S, et al.Finer mapping ofneutralizing epitope (s) on the C-terminal of Japanese encephalitis virus E-proteinexpressed in recombinant Escheriachia coli system.Vaccine, 1995,13 (16): 1515～1521.) express E albumen respectively in each and the recombinant plasmid of antigenic determinant by making up 4 kinds, detected specific antigenicity of JEV and immunogenicity, their further proof only has the fragment of one section 27 amino-acid residues (373～399) in the E albumen can induce neutralizing antibody.This research is 27 amino-acid residues to the definite minimum length of E albumen linear epitope.(Wu C J such as Wu; Huang H W; Tao M H; et al.Inductionof cross-protection against two wild-type Taiwanese isolates of Japaneseencephalitis virus using Beijing-1strain DNA vaccine.Vaccine; 2003; 21 (25-26): 3938～3945.) with the gene fragment clone of the E protein structure domain III (E292～402) of JEV CH2195LA strain isolated to prokaryotic expression carrier pET32a; express recombinant merges in E.coli; purifying merges thioredoxin (TrxD3) and freund's adjuvant or cationic-liposome mixed immunity mouse, and presentation of results TrxD3 fusion rotein can make mouse produce neutralizing antibody and immunoprotection.It is 111 amino-acid residues that this institute has analyzed the active E protein fragments length of neutralization.The linear epitope sequence of these institute's reports is long, because it is long that the length of real linear epitope is generally 6 to 12 amino-acid residues, most sequences are and the irrelevant sequence of epi-position in the fragment of 111 amino-acid residues so, not a linear epitope in this fragment perhaps, may comprise 23 or a plurality of linear epitope.Even also may comprise a plurality of linear epitopes in the fragment of 27 amino-acid residues.

Along with the development of biotechnology and information biology, protein neutral line epi-position is analyzed prevention becomes possibility.(Kutubuddin M such as Kutubuddin, Gore M M, Banerjee K, et al.Analysis ofcomputer-predicted antibody inducing epitope on Japanese encephalitis virus.ActaVirol, 1993,37 (6): be a linear neutralizing epitope 417～428.) by computer software prediction 74-CPTTGEAHNEKRAD-87.(Dewasthaly S such as Dewasthaly, Ayachit V M, Sarthi S A, et al.Monoclonal antibody raised against envelope glycoprotein peptide neutralizesJapanese encephalitis virus.Arch Virol, 2001,146:1427～1435.) epi-position by information biology software prediction Indian strain, the monoclonal antibody that has prepared the prediction epi-position is the linear neutralizing epitope of Indian strain by ELISA, indirect immunofluorescence and experimentation on animals proof 149-SENHGNYSAQVGASQ-163.This elder generation predicts to the proteantigen epi-position that by bioinformatics method again with the research method of verifying, the analysis and research work to epitope has huge promotion undoubtedly.But this method also has its weak point, though can predict some epitope by bioinformatics method, but the epi-position of this prediction must be through experimental verification, it is not real epi-position that the epi-position of prediction has many, and the method for prediction epi-position has bigger randomness, can only analyze the proteantigen epi-position to a certain extent, can not multianalysis proteantigen situation.

The present invention adopts the overlapping short peptide fusion expression technology of E albumen that covers, and E albumen is carried out the epitope mapping (Epitope Mapping) of system.The present invention carries out the linear epitope analysis to E albumen first comprehensively.Determined that at first 1～402 amino-acid residue fragment is the immunodominance determining area of E gene in the encephalitis b virus E albumen.For the epitope mapping analysis is carried out in this immunodominance zone, designed the partly overlapping small peptide of a cover, these small peptides cover 1～402 fragment total length.Each small peptide synthesizes a pair of oligonucleotide chain, and expression vector pGEX-6p-1 is inserted in the annealing back, carries out amalgamation and expression with GST.The soluble fusion rotein of expressing is with gsh affinity column chromatography purifying.Carry out Western blot and the reactive scanning of ELISA with the JEV positive serum, 9 linear epitopes have been obtained, these 9 fusion roteins are carried out immunity to mouse, obtained 9 serum that merge small peptides, by neutralization test identify E10 (73～88aa), (305～320aa) are linear neutralizing epitope to E39.Overlapping (the Kutubuddin M of neutralizing epitope E10 and former report zone 74-CPTTGEAHNEKRAD-87 wherein, Gore M M, Banerjee K, et al.Analysis of computer-predictedantibody inducing epitope on Japanese encephalitis virus.Acta Virol, 1993,37 (6): 417～428.), another neutralizing epitope E39 is for determining first.The epitope length that preliminary evaluation goes out is about 16 amino-acid residue length.For the core sequence to these epi-positions is determined, carry out the small peptide that amino-acid residue reduces from N end and C end successively at first epitope design, carry out binding analysis in the encephalitis B antiserum(antisera) behind the amalgamation and expression.Determine the core sequence of epitope, i.e. the basic sequence of this epitope can only extend and two ends all can not further reduce the sequence of any amino-acid residue to both sides.

There is apparent in view difference aspects such as virulence and blood clotting characteristic though each strain of encephalitis b virus is everlasting, and antigenic specificity is little, does not have different serotype.Encephalitis b virus on antigen with the scorching virus of black Luoshan valley head (Murray Vally Encephalitis Virus, MVEV), west nile virus (West NileVirus, WNV), St. Louis encephalitis virus (St.Louis Encephalitis Virus) has serological cross reaction, belongs to same serogroups in flaviviridae.In addition, also (DV) there is the serum cross reaction in Dengue Virus to JEV with dengue virus.These viruses all are that mosquito matchmaker property people beast suffers from transmissible disease altogether, and various viruses have also slowly broken through early stage popular territorial scope, and constantly worldwide diffusion couple human health and public health threaten.Detection timely and accurately is significant to this viroid disease of prevention and control with diagnosis, will overcome the serological cross reaction between them so, and the special diagnostic method of setting up above various virus diseases is an important job.The present invention has also carried out the epitope sequence that is identified and each flavivirus that has serological cross reaction the homology analysis of protein sequence, to also having carried out amalgamation and expression, further carry out reactivity analysis after the expression with the encephalitis b virus positive serum with other flavivirus small peptide of encephalitis b virus epitope homologous.Determined that part encephalitis b virus E proteantigen epi-position can be used for the differential diagnosis between encephalitis B and other flavivirus.With the synthetic polypeptide of the epi-position of identifying, separately or hybrid packet by elisa plate, can be used for the detection of B encephalitis virus antibody.These epitopes can be used to design recombinant vaccine, or are used for the detection of encephalitis b virus and B encephalitis virus antibody.

Summary of the invention

One of purpose of the present invention provides encephalitis b virus JEV E protein B cell antigen epitope polypeptide.

Two of purpose of the present invention is that described encephalitis b virus JEV E protein B cell antigen epitope polypeptide is applied to treatment or prevention encephalitis b virus, perhaps uses it for the reagent that is prepared into diagnosis or detects encephalitis b virus.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

A kind of antigen epitope polypeptide contains any one aminoacid sequence shown in SEQ ID NO:64, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:108, SEQ ID NO:124, the SEQ ID NO:139 in its aminoacid sequence.

In the aminoacid sequence that aforesaid antigen epitope polypeptide contained, the 7th amino acids Glu of the aminoacid sequence shown in the described SEQ ID NO:64 is replaceable to be Gln; The 7th amino acids Ser of the aminoacid sequence shown in the described SEQ ID NO:95 is replaceable to be Thr; The 6th amino acids His of the aminoacid sequence shown in the described SEQ ID NO:108 is replaceable to be Gln; The 6th amino acids Val of the aminoacid sequence shown in the described SEQ ID NO:124 is replaceable to be Ala.

Preferably, the aminoacid sequence of described antigen epitope polypeptide is any one shown in SEQ ID NO:64, SEQ IDNO:79, SEQ ID NO:95, SEQ ID NO:108, SEQ ID NO:124, the SEQ ID NO:139.

In an embodiment of the invention, preferred, the 7th amino acids Glu of the aminoacid sequence shown in the described SEQ ID NO:64 is replaceable to be Gln; The 7th amino acids Ser of the aminoacid sequence shown in the described SEQ ID NO:95 is replaceable to be Thr; The 6th amino acids His of the aminoacid sequence shown in the described SEQ ID NO:108 is replaceable to be Gln; The 6th amino acids Val of the aminoacid sequence shown in the described SEQ ID NO:124 is replaceable to be Ala.

In an embodiment of the invention, can carry out chemically modified to the N end or the C end of described antigen epitope polypeptide.Described chemically modified is the natural amination or the acetylize of polypeptide chain N end, or the carboxylated naturally or amidation of C end.

Antigen epitope polypeptide described in the present invention can obtain by solid-phase peptide synthesis is synthetic, also can prepare by genetic engineering technique, and these are all understood thoroughly by those skilled in the art.

Antigen epitope polypeptide of the present invention or its connector can be used for preparing immunogen, or with its immune animal to produce immunoprotection.Also described antigen epitope polypeptide or its connector can be prepared into the reagent that detects anti-encephalitis b virus JEV antibody or anti-encephalitis b virus JEV polypeptide antibody.

Epitope polypeptide of the present invention is prepared into immunogen or vaccine, at least comprise described one or whole epitope, these polypeptide connect, interconnect or are connected with carrier by self, and are aided with the T cell antigen epitope, comprise Th1 and/or Th2 epitope polypeptide.These T cell antigen epitopes can derive from has the immunocompetent peptide sequence of the body cell of stimulation in JEV viral protein or other animal proteinum.

Detecting encephalitis b virus JEV can be at the monoclonal antibody of E proteantigen epi-position or at the polyclonal antibody of antigen epitope polypeptide, detection method can comprise indirect ELISA, double-antibody sandwich elisa, direct immunofluorescence technic, IiT, and quick detection test paper bar immunity rete is analysed technology etc.

Detect encephalitis b virus JEV antibody, comprise that wild virus infection produces antibody, vaccine immunity produces antibody, maternal antibody, anti-JEV monoclonal antibody etc.Detection method can comprise indirect ELISA, double-antibody sandwich elisa, and quick detection test paper bar immunity rete is analysed technology etc.

The method that proteantigen B cell antigen epi-position is identified has multiple, as genetically engineered rite-directed mutagenesis antigen expressed analysis of protein method, phage random peptide library triage techniques, synthetic polypeptide detection technique etc.What this invention mainly adopted in identifying epitope is that the genetic engineering technique polypeptide merges overlapping (overlapping) expression JEV E albumen, external immune response detects the polypeptide amalgamation protein group and the sero-fast associativity of JEV is identified the epitope sequence, further shorten and mutation expression epi-position fusion rotein, identify the sequential structure of epitope; Use the exactness of synthetic polypeptide technical identification epi-position again; Prepare epitope specificity single-factor serum with epi-position fusion rotein antigen-immunized animal, analyze the epitope function.

Beneficial effect of the present invention is as follows:

1. by the antibody that can produce behind the immunogen of second antigen epitope polypeptide of the present invention design or the vaccine immunity animal body, utilize monoclonal antibody or polyclonal antibody can detect encephalitis b virus JEV or encephalitis b virus E albumen at E proteantigen epi-position at JEV.

2. antigen epitope polypeptide of the present invention is connected with carrier or self connects or interconnects; can immune animal, anti-peptide antibody that behind immune animal, produces or anti-B encephalitis virus antibody can be in vivo and in vitro in and encephalitis b virus JEV and produce immunoprotection.

3. antigen epitope polypeptide of the present invention or its connector can detect encephalitis b virus JEV antibody or anti-encephalitis b virus JEV polypeptide antibody.

Description of drawings

Fig. 1 is epitope polypeptide SEQ ID NO:64, SEQ ID NO:79, SEQ ID NO:95, SEQ IDNO:108, SEQ ID NO:124, SEQ ID NO:139 and GST amalgamation and expression albumen and anti-JEV serum Western blot detected result.Be followed successively by from left to right among the figure: M, protein molecular weight standard; 1, SEQ ID NO:64; 2, SEQ ID NO:79; 3, SEQ ID NO:95; 4, SEQ ID NO:108; 5, SEQ ID NO:124; 6, SEQ ID NO:139; 7, the GST contrast.

Fig. 2 be epitope polypeptide SEQ ID NO:124 antibody external in and the encephalitis b virus experimental result.1, the negative serum contrast; 2, anti-epitope polypeptide Seq ID No 124 serum.

Fig. 3 is epitope polypeptide sequence SEQ ID NO:64 and the comparative result of different strain JEV homologous sequences.

Fig. 4 is epitope polypeptide sequence SEQ ID NO:64 and the homologous sequence comparative result of different flaviviruss.

Fig. 5 is epitope polypeptide sequence SEQ ID NO:95 and the comparative result of different strain JEV homologous sequences.

Fig. 6 is epitope polypeptide sequence SEQ ID NO:95 and the homologous sequence comparative result of different flaviviruss.

Fig. 7 is epitope polypeptide sequence SEQ ID NO:108 and the comparative result of different strain JEV homologous sequences.

Fig. 8 is epitope polypeptide sequence SEQ ID NO:108 and the homologous sequence comparative result of different flaviviruss.

Fig. 9 is epitope polypeptide sequence SEQ ID NO:124 and the comparative result of different strain JEV homologous sequences.

Figure 10 is epitope polypeptide sequence SEQ ID NO:124 and the homologous sequence comparative result of different flaviviruss.

Embodiment

The invention will be further described below in conjunction with embodiment, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.

Embodiment 1 overlapping (over lapping) polypeptide amalgamation and expression series JEV E proteantigen polypeptide fragment is gone forward side by side and is connect ELISA screening JEV E protein B cell antigen epitope in the ranks

According to JEV (SA14-14-2 strain) E Argine Monohydrochloride sequence, design one adds up to 50 serial polypeptide, these polypeptide are overlapped, and cover JEV E albumen 1-402 amino-acid residue zone, and this zone comprises E protein structure domain I (Domain I) and domain II (Domain II).According to each bar polypeptid acid sequence, the synthetic a pair of DNA chain of design inserts expression vector pGEX-6p-1 with the DNA chain and GST carries out amalgamation and expression.The serial recombinant protein of amalgamation and expression and anti-JEV serum carry out the indirect ELISA analysis, analyze JEV E proteantigen epi-position, the results are shown in Table shown in 1.

Its idiographic flow is as follows:

DNA chain → polypeptide fusion expression vector structure → abduction delivering → expressed fusion protein affinitive layer purification → the expression product of design peptide sequence → composite coding peptide sequence and the reactive detection → analysis JEV of anti-JEV seroimmunity E proteantigen epi-position.

Table 1 polypeptide amalgamation protein and anti-JEV serum indirect ELISA reaction result

Numbering	Code name	Position (AA)	GST fusion polypeptide sequence	The OD value
					SEQ ID NO：1	E1	1～16	GST-FNCLGMGNRDFIEGAS	0.384
SEQ ID NO：2	E2	9～24	GST-RDFIEGASGATWVDLV	0.102

SEQ ID NO：3	E3	17～32	GST-GATWVDLVLEGDSCLT	0.085
					SEQ ID NO：4	E4	25～40	GST-LEGDSCLTIMANDKPT	0.087
SEQ ID NO：5	E5	33～48	GST-IMANDKPTLDVRMINI	0.090
					SEQ ID NO：6	E6	41～56	GST-LDVRMINIEASQLAEV	0.010
SEQ ID NO：7	E7	49～64	GST-EASQLAEVRSYCYHAS	0.078
					SEQ ID NO：8	E8	57～72	GST-RSYCYHASVTDISTVA	0.086
SEQ ID NO：9	E9	65～80	GST-VTDISTVARCPTTGEA	0.098
					SEQ ID NO：10	E10	73～88	GST-RCPTTGEAHNEKRADS	0.405
SEQ ID NO：11	E11	81～96	GST-HNEKRADSSYVCKQGF	0.550
					SEQ ID NO：12	E12	89～104	GST-SYVCKQGFTDRGWGNG	0.074
SEQ ID NO：13	E13	97～112	GST-TDRGWGNGCGFFGKGS	0.103
					SEQ ID NO：14	E14	105～120	GST-CGFFGKGSIDTCAKFS	0.104
SEQ ID NO：15	E15	113～128	GST-IDTCAKFSCTSKAIGR	0.085
					SEQ ID NO：16	E16	121～136	GST-CTSKAIGRTIQPENIK	0.096
SEQ ID NO：17	E17	129～144	GST-TIQPENIKYKVGIFVH	0.097
					SEQ ID NO：18	E18	137～152	GST-YKVGIFVHGTTTSENH	0.084
SEQ ID NO：19	E19	145～160	GST-GTTTSENHGNYSAQVG	0.560
					SEQ ID NO：20	E20	153～168	GST-GNYSAQVGASQAAKFT	0.097
SEQ ID NO：21	E21	161～176	GST-ASQAAKFTVTPNAPSV	0.112
					SEQ ID NO：22	E22	169～184	GST-VTPNAPSVALKLGDYG	0.086
SEQ ID NO：23	E23	177～192	GST-ALKLGDYGEVTLDCEP	0.087
					SEQ ID NO：24	E24	185～200	GST-EVTLDCEPRSGLNTEA	0.098
SEQ ID NO：25	E25	193～208	GST-RSGLNTEAFYVMTVGS	0.100
					SEQ ID NO：26	E26	201～216	GST-FYVMTVGSKSFLVHRE	0.088
SEQ ID NO：27	E27	209～224	GST-KSFLVHREWFHDLALP	0.089
					SEQ ID NO：28	E28	217～232	GST-WFHDLALPWTSPSSTA	0.105
SEQ ID NO：29	E29	225～240	GST-WTSPSSTAWRNRELLM	0.117
					SEQ ID NO：30	E30	233～248	GST-WRNRELLMEFEGAHAT	0.079
SEQ ID NO：31	E31	241～256	GST-EFEGAHATKQSVVALG	0.086
					SEQ ID NO：32	E32	249～264	GST-KQSVVALGSQEGGLHH	0.110

SEQ ID NO：33	E33	257～272	GST-SQEGGLHHALAGAIVV	0.460
					SEQ ID NO：34	E34	265～280	GST-ALAGAIVVEYSSSVML	0.104
SEQ ID NO：35	E35	273～288	GST-EYSSSVMLTSGHLKCR	0.105
					SEQ ID NO：36	E36	281～296	GST-TSGHLKCRLKMDKLAL	0.094
SEQ ID NO：37	E37	289～304	GST-LKMDKLALKGTTYGMC	0.086
					SEQ ID NO：38	E38	297～312	GST-KGTTYGMCTEKFSFAK	0.097
SEQ ID NO：39	E39	305～320	GST-TEKFSFAKNPVDTGHG	0.450
					SEQ ID NO：40	E40	313～328	GST-NPVDTGHGTVVIELSY	0.087
SEQ ID NO：41	E41	321～336	GST-TVVIELSYSGSDGPCK	0.078
					SEQ ID NO：42	E42	329～344	GST-SGSDGPCKIPIVSVAS	0.096
SEQ ID NO：43	E43	337～352	GST-IPIVSVASLNDMTPVG	0.093
					SEQ ID NO：44	E44	345～360	GST-LNDMTPVGRLVTVNPF	0.09
SEQ ID NO：45	E45	353～368	GST-RLVTVNPFVATSSANS	0.377
					SEQ ID NO：46	E46	361～376	GST-VATSSANSKVLVEMEP	0.098
SEQ ID NO：47	E47	369～384	GST-KVLVEMEPPFGDSYIV	0.385
					SEQ ID NO：48	E48	377～392	GST-PFGDSYIVVGRGDKQI	0.390
SEQ ID NO：49	E49	385～400	GST-VGRGDKQINHHWHKAG	0.430
					SEQ ID NO：50	E50	393～408	GST-NHHWHKAGSTLGKAFS	0.103
	Contrast		GST	0.118

Annotate: envelope antigen is respectively polypeptide amalgamation protein, and GST is the contrast envelope antigen, and antigen coated concentration is 10 μ g/ml; Anti-JEV serum is 200 times of dilutions, and 450nm wavelength OD value is read in the tmb substrate colour developing.

Interpretation of result shows that the epitope sequence that preliminary evaluation goes out comprises SEQ ID NO:1, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:19, SEQ ID NO:33, SEQ ID NO:39, SEQ IDNO:45, SEQ ID NO:48 and SEQ ID NO:49.

The core sequence location of embodiment 2 epitopes

The epitope sequence that goes out according to preliminary evaluation among the embodiment 1, further design the small segment polypeptide, or design is held to N from C and is reduced the serial polypeptide of an amino-acid residue successively and hold the serial polypeptide that reduces an amino-acid residue to C end successively from N, carrying out Western blot with the JEV positive serum behind the amalgamation and expression analyzes, determine the core sequence of this epi-position when two ends are reduced to the polypeptide amalgamation protein forfeiture with JEV positive serum bonded ability respectively, reaction result is shown in Table 2.

Table 2 polypeptide amalgamation protein and the indirect Western blot of anti-JEV serum reaction result

Numbering	Code name	GST fusion polypeptide sequence	Western blot detected result
				SEQ ID NO：1	E1	FNCLGMGNRDFIEGAS	+++
SEQ ID NO：51	E1-1	FNCLGMGNRDFIEGA	+++
				SEQ ID NO：52	E1-2	FNCLGMGNRDFIEG	+++
SEQ ID NO：53	E1-3	FNCLGMGNRDFIE	-
				SEQ ID NO：54	E1-4	FNCLGMGNRDFI	-
SEQ ID NO：55	E1-5	FNCLGMGNRDF	-
				SEQ ID NO：56	E1-6	FNCLGMGNRD	-
SEQ ID NO：57	E1-7	FNCLGMGNR	-
				SEQ ID NO：58	E1-8	FNCLGMGN	-
SEQ ID NO：59	E1-9	NCLGMGNRDFIEG	+++
				SEQ ID NO：60	E1-10	CLGMGNRDFIEG	+++
SEQ ID NO：61	E1-11	LGMGNRDFIEG	+++
				SEQ ID NO：62	E1-12	GMGNRDFIEG	+++
SEQ ID NO：63	E1-13	MGNRDFIEG	+++
				SEQ ID NO：64	E1-14	GNRDFIEG	+++
SEQ ID NO：65	E1-15	NRDFIEG	-
				SEQ ID NO：66	E1-16	RDFIEG	-
SEQ ID NO：10	E10	RCPTTGEAHNEKRADS	+++
				SEQ ID NO：67	E10-1	RCPTTGEA	-
SEQ ID NO：68	E10-2	HNEKRADS	-
				SEQ ID NO：69	E10-3	PTTGEAHNEKRA	+++
SEQ ID NO：70	E10-4	TGEAHNEK	++
				SEQ ID NO：11	E11	HNEKRADSSYVCKQGF	+++
SEQ ID NO：71	E11-1	SYVCKQGF	-
				SEQ ID NO：72	E11-2	EKRADSSYVCKQ	+++

SEQ ID NO：73	E11-3	RADSSYVC	-
				SEQ ID NO：74	E11-4	EKRADSSYVCKQ	+++
SEQ ID NO：75	E11-5	EKRADSSYVCK	+++
				SEQ ID NO：76	E11-6	EKRADSSYVC	+++
SEQ ID NO：77	E11-7	EKRADSSYV	+++
				SEQ ID NO：78	E11-8	EKRADSSY	+++
SEQ ID NO：79	E11-9	EKRADSS	+++
				SEQ ID NO：80	E11-10	EKRADS	-
SEQ ID NO：81	E11-11	KRADSSY	-
				SEQ ID NO：82	E11-12	RADSSY	-
SEQ ID NO：19	E19	GTTTSENHGNYSAQVG	+++
				SEQ ID NO：83	E19-1	GTTTSENHGNYSAQV	+++
SEQ ID NO：84	E19-2	GTTTSENHGNYSAQ	+++
				SEQ ID NO：85	E19-3	GTTTSENHGNYSA	+++
SEQ ID NO：86	E19-4	GTTTSENHGNYS	+++
				SEQ ID NO：87	E19-5	GTTTSENHGNY	-
SEQ ID NO：88	E19-6	GTTTSENHGN	-
				SEQ ID NO：89	E19-7	GTTTSENHG	-
SEQ ID NO：90	E19-8	GTTTSENH	-
				SEQ ID NO：91	E19-9	TTTSENHGNYS	+++
SEQ ID NO：92	E19-10	TTSENHGNYS	+++
				SEQ ID NO：93	E19-11	TSENHGNYS	+++
SEQ ID NO：94	E19-12	SENHGNYS	+++
				SEQ ID NO：95	E19-13	ENHGNYS	+++
SEQ ID NO：96	E19-14	NHGNYS	-
				SEQ ID NO：97	E19-15	HGNYS	-
SEQ ID NO：98	E19-16	GNYS	-
				SEQ ID NO：33	E33	SQEGGLHHALAGAIVV	+++
SEQ ID NO：99	E33-1	SQEGGLHHALAGAIV	+++
				SEQ ID NO：100	E33-2	SQEGGLHHALAGAI	+++

SEQ ID NO：101	E33-3	SQEGGLHHALAGA	+++
				SEQ ID NO：102	E33-4	SQEGGLHHALAG	+++
SEQ ID NO：103	E33-5	SQEGGLHHALA	+++
				SEQ ID NO：104	E33-6	SQEGGLHHAL	+++
SEQ ID NO：105	E33-7	SQEGGLHHA	+++
				SEQ ID NO：106	E33-8	SQEGGLHH	-
SEQ ID NO：107	E33-9	QEGGLHHA	+++
				SEQ ID NO：108	E33-10	EGGLHHA	+++
SEQ ID NO：109	E33-11	GGLHHA	-
				SEQ ID NO：110	E33-12	GLHHA	-
SEQ ID NO：111	E33-13	LHHA	-
				SEQ ID NO：39	E39	TEKFSFAKNPVDTGHG	+++
SEQ ID NO：112	E39-1	TEKFSFAKNPVDTGH	+++
				SEQ ID NO：113	E39-2	TEKFSFAKNPVDTG	+++
SEQ ID NO：114	E39-3	TEKFSFAKNPVDT	+++
				SEQ ID NO：115	E39-4	TEKFSFAKNPVD	+++
SEQ ID NO：116	E39-5	TEKFSFAKNPV	-
				SEQ ID NO：117	E39-6	TEKFSFAKNP	-
SEQ ID NO：118	E39-7	TEKFSFAKN	-
				SEQ ID NO：119	E39-8	TEKFSFAK	-
SEQ ID NO：120	E39-9	EKFSFAKNPVD	+++
				SEQ ID NO：121	E39-10	KFSFAKNPVD	+++
SEQ ID NO：122	E39-11	FSFAKNPVD	+++
				SEQ ID NO：123	E39-12	SFAKNPVD	+++
SEQ ID NO：124	E39-13	FAKNPVD	+++
				SEQ ID NO：125	E39-14	AKNPVD	-
SEQ ID NO：126	E39-15	KNPVD	-
				SEQ ID NO：127	E39-16	NPVD	-
SEQ ID NO：45	E45	RLVTVNPFVATSSANS	+++
				SEQ ID NO：128	E45-1	VTVNPFVATSSA	+++

SEQ ID NO：129	E45-2	VNPFVATS	+++
				SEQ ID NO：130	E45-3	PFVA	++
SEQ ID NO：48	E48	PFGDSYIVVGRGDKQI	+++
				SEQ ID NO：131	E48-1	PFGDSYIV	+++
SEQ ID NO：132	E48-2	VGRGDKQI	+++
				SEQ ID NO：133	E48-3	GDSYIVVGRGDK	-
SEQ ID NO：134	E48-4	SYIVVGRG	-
				SEQ ID NO：49	E49	VGRGDKQINHHWHKAG	+++
SEQ ID NO：135	E49-1	NHHWHKAG	-
				SEQ ID NO：136	E49-2	RGDKQINHHWHK	-
SEQ ID NO：137	E49-3	DKQINHHW	-
				SEQ ID NO：132	E48-2	VGRGDKQI	+++
SEQ ID NO：138	E49-4	VGRGDKQ	+++
				SEQ ID NO：139	E49-5	VGRGDK	+++
SEQ ID NO：140	E49-6	VGRGD	-
				SEQ ID NO：141	E49-7	GRGDKQI	-
SEQ ID NO：142	E49-8	RGDKQI	-
				SEQ ID NO：143	E49-9	GDKQI	-
	Contrast	GST	-

Annotate: detect the gst fusion protein that antigen is respectively polypeptide shown in the table, GST is contrast antigen, and anti-JEV serum is 200 times of dilutions.

Interpretation of result shows that the epitope core sequence of determining is SEQ ID NO:64 (E1-14), SEQID NO:79 (E11-9), SEQ ID NO:95 (E19-13), SEQ ID NO:108 (E33-10), SEQ ID NO:124 (E39-13), SEQ ID NO:139 (E49-5).

The mutation analysis of embodiment 3 epitope core sequences

The epitope core sequence of determining among the embodiment 2 and different strain encephalitis B E protein sequences relatively and with black Luoshan valley head scorching viral (MVEV), west nile virus (WNV), its virus (UsutuVirus of outstanding Soviet Union, UV), elder brother Tianjin virus (Kunjin virus, KUN) and dengue virus (Dengue Virus DV) waits flavivirus E albumen to carry out homology analysis relatively.

Relatively according to epitope core sequence and other the viral homology determined among the embodiment 2, get homology earlier greater than 50% homologous sequence small peptide, be SEQ ID NO:64, SEQ ID NO:95, SEQ IDNO:108 and SEQ ID NO:124, it is carried out Western blot behind the GST amalgamation and expression analyze reactivity with the JEV positive serum, reaction result is shown in Table 3.

Table 3 polypeptide amalgamation protein and anti-JEV serum Western blot reaction result

Annotate: detect the gst fusion protein that antigen is respectively polypeptide shown in the table, GST is contrast antigen, and anti-JEV serum is 200 times of dilutions.

Interpretation of result shows that epi-position SEQ ID NO:64 (E1-14), SEQ ID NO:95 (E19-13), SEQ ID NO:108 (E33-10) have virus-specific, and the homologous sequence of other flavivirus can not be discerned by the JEV positive serum.

And, described sequence SEQ ID NO:64, its 7th amino acids Glu is replaceable to be Gln, the aminoacid sequence after the replacement is SEQ ID NO:144;

Described sequence SEQ ID NO:95, its 7th amino acids Ser is replaceable to be Thr, the aminoacid sequence after the replacement is SEQ ID NO:151;

Described sequence SEQ ID NO:108, its 6th amino acids His is replaceable to be Gln, the aminoacid sequence after the replacement is SEQ ID NO:157;

Described sequence SEQ ID NO:124, its 6th amino acids Val is replaceable to be Ala, the aminoacid sequence after the replacement is SEQ ID NO:164.

Synthesizing of embodiment 4 encephalitis b virus JEV E protein B cell antigen epitopes

According to the aminoacid sequence of JEV E protein B cell antigen epitope, use the solid-phase polypeptide synthetic technology, adopt automatic Peptide synthesizer or artificial polypeptide synthesis method.Solid-phase resin adopts amino acid Wang resin or other resin of Fmoc protection, and resin according to the aminoacid sequence order, adds the amino acid of Fmoc protection after DMF swelling, piperidine glue Fmoc protection, exist at HBTU and carry out acylation reaction under the situation.Through washing, add deputy Fmoc-amino acid again and carry out acylation reaction after acylation reaction is finished, and washing.So circulation is super initial to N-terminal from the peptide sequence C-terminal, synthetic in order complete polypeptide chain.After synthetic the finishing, choose suitable reagent with being connected and precipitating the TFA polypeptide of TFA method cracking peptide chain and resin with cold diethyl ether according to component peptide chain amino acid difference, standby after desalting and purifying, LC-MS and HPLC Analysis and Identification.In entire synthesis process, can use the performance of Kaiser method or TMBS method test reaction after each amino acid acylation reaction.For the ease of epitope polypeptide is connected with carrier proteins, when synthesizing polypeptide, can add a halfcystine at the N-terminal of peptide sequence.

According to the method described above, the polypeptide fragment that has synthesized aminoacid sequence shown in SEQ ID NO:64, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:108, SEQ ID NO:124, the SEQ ID NO:139 respectively.

The chemically modified of embodiment 5 encephalitis b virus JEV E protein B cell antigen epitopes

According to the method synthetic polypeptide fragment of embodiment 4, its N-terminal is that nature is amido modified, and C-terminal is the nature carboxyl modified.

N-terminal acetylation modification method: the polypeptide according to above introduction is blended into last Fmoc protection amino acid coupling end, and carries out the removal of N-terminal Fmoc blocking group, carries out following operation then.Get 150 μ l diacetyl oxides and 20 μ L EIPEA are dissolved among the 4.8ml DMF, thorough mixing also cools off in ice bath.Then above mixed solution is added in the polypeptide building-up reactions pipe and react 5min.Use 5ml DMF and methylene dichloride (DCM) to wash again successively 2 times, each 5min.Nitrogen dries up the back and carries out removal, cracking, purifying and the evaluation of side chain protected group according to ordinary method.

C-terminal amidation modifying method: should select resin for use when the amidated polypeptide of C-terminal is synthetic is the Rink resin.The reply resin begins to carry out synthesis program from C-terminal first amino acids then with DMF swelling 20min when carrying out synthesis program, the same Fmoc-wang resin synthetic method of crossing.

The coupling of embodiment 6 encephalitis B JEV E protein B cell antigen epitopes and carrier proteins KLH and BSA:

Carrier proteins KLH or BSA 4mg are dissolved in PBS (pH7.2) damping fluid that 0.5ml contains 5mM EDTA, add 1.0mg linking agent Sulfo-SMCC, and fully 30min is hatched incubated at room 60min or 37 ℃ in the dissolving back.Use the carrier proteins that Sephadex G-25 post or dialysis process desalting and purifying Sulfo-SMCC handle, and with standby behind the BCA method mensuration protein content.

Take by weighing synthetic epitope polypeptide 4mg, add 0.5ml double distilled water dissolving polypeptide, the polypeptide after will dissolving then mixes with the carrier proteins that Sulfo-SMCC handles, and hatches 2 hours or reacts and spend the night for 4 ℃, and packing is standby.

Embodiment 7 encephalitis b virus JEV E protein B cell antigen epitope oxidations realize the connection of self

The polypeptide that contains halfcystine can form self and connect by the oxidizing reaction of sulfydryl.The oxidizing reaction of using the DMSO mediation generally speaking realizes connecting, and according to the acid-basicity of polypeptide, oxidizing reaction can be carried out under little acid or slight alkalinity condition.

Oxidizing reaction under the slightly acidic environment: with the acetate dissolution polypeptide of proper concn, the concentration that makes polypeptide is in the 0.5-1.5mM scope, the final concentration of acetic acid is no more than 5%, the pH that adjusts polypeptide solution with (NH4) 2CO3 is about 6.0, the DMSO that adds 10-20%, 25 ℃ of reaction 5-25h, the carrying out degree and can monitor of oxidizing reaction by HPLC.Then, be moving phase with 1%TFA water and acetonitrile, preparation type reversed-phase HPLC purifying oxidisability polypeptide.

Oxidizing reaction under the slight alkalinity environment: use the 0.01M phosphate buffered saline buffer, pH7.5, dissolving polypeptide add concentration DMSO and spend the night to 1%, 25 ℃ of reaction of final concentration to final concentration 1.0mM, the carrying out degree and can monitor by HPLC of oxidizing reaction.Be moving phase with 1%TFA water and acetonitrile then, preparation type reversed-phase HPLC purifying oxidisability polypeptide.

Embodiment 8 encephalitis b virus JEV E protein B cell antigen epitope indirect ELISAs detect the anti-JEV antibody of pig

Connector mixture with JEV E protein B cell antigen epitope SEQ ID NO:64, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:108, SEQ ID NO:124, the synthetic epitope polypeptide of SEQ ID NO:139 and carrier is an envelope antigen, and bag is by 96 hole polystyrene enzyme plates.Use pH9.6,0.1M carbonate buffer solution dilution antigen to final concentration is 10 μ g/ml, adds in the enzyme plate by 100 μ l/ holes, and 4 ℃ of bags are spent the night.Use PBST (PBS+0.05%Tween) detersive enzyme target 3 times then; The PBST sealase target that contains 1%BSA, 37 ℃ of sealing 2h, plate is washed 3 times with washing lotion PBST in sealing back, is used for detection or-20 ℃ immediately and deposits standby.

Antibody test schedule of operation: add porcine blood serum to be detected (100 times of dilutions of qualitative detection serum, antibody titer detects serum and carries out doubling dilution, set up positive serum contrast and negative serum contrast and the blank of increase serum not simultaneously), hatch 1h for 37 ℃, PBST washing lotion washing 3 times, each 3 minutes; Add horseradish peroxidase-labeled goat-anti pig IgG (Goat-anti-pig IgG-HRP), hatch 1h for 37 ℃, PBST washing lotion washing 4 times, each 3 minutes; Add HRP chromogenic substrate (TMB developer), incubated at room 5-15 minute observation color reaction; Fully after the colour developing, add the reaction of 2M sulfuric acid color development stopping; Measure the light absorption value of 450nm wavelength with microplate reader; Result of determination.The results are shown in Table shown in 4.

Table 4 mixed polypeptide bag is detected the anti-JEV antibody of pig by indirect ELISA

Annotate: envelope antigen is the mixture of epitope SEQ ID NO:64, SEQ ID NO:79, SEQ ID NO:95, SEQID NO:108, the synthetic polypeptide of SEQ ID NO:124, SEQ ID NO:139 and BSA connector, 10 μ g/m1.It is 200 that this positive serum is tired.

During result of determination: blank and negative serum hole light absorption value are less than or equal to 0.2, and positive serum control wells light absorption value is effective greater than 0.4 o'clock result; Calculate P/N value=(detection hole OD value-blank hole OD value)/(negative serum OD value-blank hole OD value), the P/N value be equal to or greater than 2 o'clock positive; Maximum dilution multiple with reacting positive serum is the antibody titer of this sample serum.

Embodiment 9 encephalitis b virus JEV E protein B cell antigen epitope indirect ELISAs detect the anti-JEV antibody of people

Implementation method is with embodiment 8, and serum to be checked, negative control sera and positive control serum are human serum; Two anti-are horseradish peroxidase-labeled goat anti-human igg (Goat-anti human IgG-HRP).

Embodiment 10 encephalitis b virus JEV E protein B cell antigen epitope indirect ELISAs detect the anti-JEV antibody of horse

Implementation method is with embodiment 8, and serum to be checked, negative control sera and positive control serum are horse serum; Two anti-are horseradish peroxidase-labeled goat-anti horse IgG (Goat-anti horse IgG-HRP).

Embodiment 11 encephalitis b virus JEV E protein B cell antigen epitope Dot-ELISA detect the anti-JEV antibody of pig

Mixture with the connector of JEV E protein B cell antigen epitope SEQ ID NO:64, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:108, SEQ ID NO:124, SEQ ID NO:139 epitope polypeptide and carrier is an envelope antigen, is contrast antigen with BSA.The envelope antigen of on nitrocellulose filter, fixing a point, 2 μ g/ points, 37 ℃ of fixing 30min, PBST washing 6 times, 4 ℃ of sealings are spent the night among the 2%BSA, promptly can be used for antibody test after the PBST washing 6 times.

When detecting antibody, serum sample to be detected is suitably diluted the back hatch 30min, PBST washing 6 times, adding horseradish peroxidase-labeled goat-anti pig IgG (Goat-anti-pigIgG-HRP) in 37 ℃ with antigen coated film, hatch 1h for 37 ℃, PBST washing lotion washing 6 times; Add the colour developing of AEC or DAB substrate, the room temperature lucifuge is hatched 10min observation color reaction.

When the result judges, present the red-brown color reaction with the envelope antigen point, contrast antigen point color reaction does not the occur, and the person is positive.

Embodiment 12 encephalitis b virus JEV E protein B cell antigen epitope Dot-ELISA detect the anti-JEV antibody of people

Implementation method is with embodiment 11, and serum to be checked is human serum; Two anti-are horseradish peroxidase-labeled goat anti-human igg (Goat-anti human IgG-HRP).

Embodiment 13 encephalitis b virus JEV E protein B cell antigen epitope Dot-ELISA detect the anti-JEV antibody of horse

Implementation method is with embodiment 11, and serum to be checked is horse serum; Two anti-are horseradish peroxidase-labeled goat-anti horse IgG (Goat-anti horse IgG-HRP).

Embodiment 14 immunogenic preparation of encephalitis b virus JEV E protein B cell antigen epitope and immunization experiments

Encephalitis b virus JEV E protein B cell antigen epitope immunogen can be the synthetic antigen epitope polypeptide, antigen epitope polypeptide self connector, and antigen epitope polypeptide and carrier connector, epitope merges epi-position albumen etc.Be immunogen with antigen epitope polypeptide SEQ ID NO:64, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:108, SEQ ID NO:124, SEQ ID NO:139 and BSA connector in the present embodiment; First immunisation immunogen Freund's complete adjuvant and antigen emulsification, animalcule immunizing antigen amount is every of 100 μ g; Two or three when exempting from Freund's incomplete adjuvant and antigen emulsification, animalcule immunizing antigen amount is that every of 50-100 μ g carries out immunity.Immunization route can be subcutaneous injection, intradermal injection or intramuscular injection.Each time duration of immunity is two weeks at interval.Antibody horizontal is detected in twice back of booster immunization.

Test-results sees Table 5 respectively, table 6, table 7, table 8, table 9, table 10.

Table 5 mouse anti epitope polypeptide E1 antibody indirect ELISA is tired

Annotate: envelope antigen is antigen epitope polypeptide SEQ NO:64,10 μ g/ml.

Table 6 mouse anti epitope polypeptide E11 antibody indirect ELISA is tired

Annotate: envelope antigen is antigen epitope polypeptide SEQ NO:79,10 μ g/ml.

Table 7 mouse anti epitope polypeptide E19 antibody indirect ELISA is tired

Annotate: envelope antigen is antigen epitope polypeptide SEQ NO:95,10 μ g/ml.

Table 8 mouse anti epitope polypeptide E33 antibody indirect ELISA is tired

Annotate: envelope antigen is antigen epitope polypeptide SEQ NO:108,10 μ g/ml.

Table 9 mouse anti epitope polypeptide E39 antibody indirect ELISA is tired

Annotate: envelope antigen is antigen epitope polypeptide SEQ NO:124,10 μ g/ml.

Table 10 mouse anti epitope polypeptide E49 antibody indirect ELISA is tired

Annotate: envelope antigen is antigen epitope polypeptide SEQ NO:139,10 μ g/ml.

The above only is preferred embodiment of the present invention, only is illustrative for the purpose of the present invention, and nonrestrictive.Those skilled in the art is understood, and can carry out many changes to it in the spirit and scope that claim of the present invention limited, revise, even, equivalence, but all will fall within the scope of protection of the present invention.

One group of antigen epitope polypeptide sequence table

SEQUENCE LISTING

＜110〉Harbin Veterinary Medicine Inst., China Academy of Agriculture

＜120〉one group of antigen epitope polypeptide and application thereof

<130>KLPI090

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<213>Japanese encephalitis virus

<400>40

Asn Pro Val Asp Thr Gly His Gly Thr Val Val Ile Glu Leu Ser Tyr

1 5 10 15

<210>41

<211>16

<212>PRT

<213>Japanese encephalitis virus

<400>41

Thr Val Val Ile Glu Leu Ser Tyr Ser Gly Ser Asp Gly Pro Cys Lys

1 5 10 15

<210>42

<211>16

<212>PRT

<213>Japanese encephalitis virus

<400>42

Ser Gly Ser Asp Gly Pro Cys Lys Ile Pro Ile Val Ser Val Ala Ser

1 5 10 15

<210>43

<211>16

<212>PRT

<213>Japanese encephalitis virus

<400>43

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

1 5 10 15

<210>44

<211>16

<212>PRT

<213>Japanese encephalitis virus

<400>44

Leu Asn Asp Met Thr Pro Val Gly Arg Leu Val Thr Val Asn Pro Phe

1 5 10 15

<210>45

<211>16

<212>PRT

<213>Japanese encephalitis virus

<400>45

Arg Leu Val Thr Val Asn Pro Phe Val Ala Thr Ser Ser Ala Asn Ser

1 5 10 15

<210>46

<211>16

<212>PRT

<213>Japanese encephalitis virus

<400>46

Val Ala Thr Ser Ser Ala Asn Ser Lys Val Leu Val Glu Met Glu Pro

1 5 10 15

<210>47

<211>16

<212>PRT

<213>Japanese encephalitis virus

<400>47

Lys Val Leu Val Glu Met Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val

1 5 10 15

<210>48

<211>16

<212>PRT

<213>Japanese encephalitis virus

<400>48

Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg Gly Asp Lys Gln Ile

1 5 10 15

<210>49

<211>16

<212>PRT

<213>Japanese encephalitis virus

<400>49

Val Gly Arg Gly Asp Lys Gln Ile Asn His His Trp His Lys Ala Gly

1 5 10 15

<210>50

<211>16

<212>PRT

<213>Japanese encephalitis virus

<400>50

Asn His His Trp His Lys Ala Gly Ser Thr Leu Gly Lys Ala Phe Ser

1 5 10 15

<210>51

<211>15

<212>PRT

<213>Japanese encephalitis virus

<400>51

Phe Asn Cys Leu Gly Met Gly Asn Arg Asp Phe Ile Glu Gly Ala

1 5 10 15

<210>52

<211>14

<212>PRT

<213>Japanese encephalitis virus

<400>52

Phe Asn Cys Leu Gly Met Gly Asn Arg Asp Phe Ile Glu Gly

1 5 10

<210>53

<211>13

<212>PRT

<213>Japanese encephalitis virus

<400>53

Phe Asn Cys Leu Gly Met Gly Asn Arg Asp Phe Ile Glu

1 5 10

<210>54

<211>12

<212>PRT

<213>Japanese encephalitis virus

<400>54

Phe Asn Cys Leu Gly Met Gly Asn Arg Asp Phe Ile

1 5 10

<210>55

<211>11

<212>PRT

<213>Japanese encephalitis virus

<400>55

Phe Asn Cys Leu Gly Met Gly Asn Arg Asp Phe

1 5 10

<210>56

<211>10

<212>PRT

<213>Japanese encephalitis virus

<400>56

Phe Asn Cys Leu Gly Met Gly Asn Arg Asp

1 5 10

<210>57

<211>9

<212>PRT

<213>Japanese encephalitis virus

<400>57

Phe Asn Cys Leu Gly Met Gly Asn Arg

1 5

<210>58

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>58

Phe Asn Cys Leu Gly Met Gly Asn

1 5

<210>59

<211>13

<212>PRT

<213>Japanese encephalitis virus

<400>59

Asn Cys Leu Gly Met Gly Asn Arg Asp Phe Ile Glu Gly

1 5 10

<210>60

<211>12

<212>PRT

<213>Japanese encephalitis virus

<400>60

Cys Leu Gly Met Gly Asn Arg Asp Phe Ile Glu Gly

1 5 10

<210>61

<211>11

<212>PRT

<213>Japanese encephalitis virus

<400>61

Leu Gly Met Gly Asn Arg Asp Phe Ile Glu Gly

1 5 10

<210>62

<211>10

<212>PRT

<213>Japanese encephalitis virus

<400>62

Gly Met Gly Asn Arg Asp Phe Ile Glu Gly

1 5 10

<210>63

<211>9

<212>PRT

<213>Japanese encephalitis virus

<400>63

Met Gly Asn Arg Asp Phe Ile Glu Gly

1 5

<210>64

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>64

Gly Asn Arg Asp Phe Ile Glu Gly

1 5

<210>65

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>65

Asn Arg Asp Phe Ile Glu Gly

1 5

<210>66

<211>6

<212>PRT

<213>Japanese encephalitis virus

<400>66

Arg Asp Phe Ile Glu Gly

1 5

<210>67

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>67

Arg Cys Pro Thr Thr Gly Glu Ala

1 5

<210>68

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>68

His Asn Glu Lys Arg Ala Asp Ser

1 5

<210>69

<211>12

<212>PRT

<213>Japanese encephalitis virus

<400>69

Pro Thr Thr Gly Glu Ala His Asn Glu Lys Arg Ala

1 5 10

<210>70

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>70

Thr Gly Glu Ala His Asn Glu Lys

1 5

<210>71

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>71

Ser Tyr Val Cys Lys Gln Gly Phe

1 5

<210>72

<211>12

<212>PRT

<213>Japanese encephalitis virus

<400>72

Glu Lys Arg Ala Asp Ser Ser Tyr Val Cys Lys Gln

1 5 10

<210>73

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>73

Arg Ala Asp Ser Ser Tyr Val Cys

1 5

<210>74

<211>12

<212>PRT

<213>Japanese encephalitis virus

<400>74

Glu Lys Arg Ala Asp Ser Ser Tyr Val Cys Lys Gln

1 5 10

<210>75

<211>11

<212>PRT

<213>Japanese encephalitis virus

<400>75

Glu Lys Arg Ala Asp Ser Ser Tyr Val Cys Lys

1 5 10

<210>76

<211>10

<212>PRT

<213>Japanese encephalitis virus

<400>76

Glu Lys Arg Ala Asp Ser Ser Tyr Val Cys

1 5 10

<210>77

<211>9

<212>PRT

<213>Japanese encephalitis virus

<400>77

Glu Lys Arg Ala Asp Ser Ser Tyr Val

1 5

<210>78

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>78

Glu Lys Arg Ala Asp Ser Ser Tyr

1 5

<210>79

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>79

Glu Lys Arg Ala Asp Ser Ser

1 5

<210>80

<211>6

<212>PRT

<213>Japanese encephalitis virus

<400>80

Glu Lys Arg Ala Asp Ser

1 5

<210>81

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>81

Lys Arg Ala Asp Ser Ser Tyr

1 5

<210>82

<211>6

<212>PRT

<213>Japanese encephalitis virus

<400>82

Arg Ala Asp Ser Ser Tyr

1 5

<210>83

<211>15

<212>PRT

<213>Japanese encephalitis virus

<400>83

Gly Thr Thr Thr Ser Glu Asn His Gly Asn Tyr Ser Ala Gln Val

1 5 10 15

<210>84

<211>14

<212>PRT

<213>Japanese encephalitis virus

<400>84

Gly Thr Thr Thr Ser Glu Asn His Gly Asn Tyr Ser Ala Gln

1 5 10

<210>85

<211>13

<212>PRT

<213>Japanese encephalitis virus

<400>85

Gly Thr Thr Thr Ser Glu Asn His Gly Asn Tyr Ser Ala

1 5 10

<210>86

<211>12

<212>PRT

<213>Japanese encephalitis virus

<400>86

Gly Thr Thr Thr Ser Glu Asn His Gly Asn Tyr Ser

1 5 10

<210>87

<211>11

<212>PRT

<213>Japanese encephalitis virus

<400>87

Gly Thr Thr Thr Ser Glu Asn His Gly Asn Tyr

1 5 10

<210>88

<211>10

<212>PRT

<213>Japanese encephalitis virus

<400>88

Gly Thr Thr Thr Ser Glu Asn His Gly Asn

1 5 10

<210>89

<211>9

<212>PRT

<213>Japanese encephalitis virus

<400>89

Gly Thr Thr Thr Ser Glu Asn His Gly

1 5

<210>90

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>90

Gly Thr Thr Thr Ser Glu Asn His

1 5

<210>91

<211>11

<212>PRT

<213>Japanese encephalitis virus

<400>91

Thr Thr Thr Ser Glu Asn His Gly Asn Tyr Ser

1 5 10

<210>92

<211>10

<212>PRT

<213>Japanese encephalitis virus

<400>92

Thr Thr Ser Glu Asn His Gly Asn Tyr Ser

1 5 10

<210>93

<211>9

<212>PRT

<213>Japanese encephalitis virus

<400>93

Thr Ser Glu Asn His Gly Asn Tyr Ser

1 5

<210>94

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>94

Ser Glu Asn His Gly Asn Tyr Ser

1 5

<210>95

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>95

Glu Asn His Gly Asn Tyr Ser

1 5

<210>96

<211>6

<212>PRT

<213>Japanese encephalitis virus

<400>96

Asn His Gly Asn Tyr Ser

1 5

<210>97

<211>5

<212>PRT

<213>Japanese encephalitis virus

<400>97

His Gly Asn Tyr Ser

1 5

<210>98

<211>4

<212>PRT

<213>Japanese encephalitis virus

<400>98

Gly Asn Tyr Ser

1

<210>99

<211>15

<212>PRT

<213>Japanese encephalitis virus

<400>99

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

1 5 10 15

<210>100

<211>14

<212>PRT

<213>Japanese encephalitis virus

<400>100

Ser Gln Glu Gly Gly Leu His His Ala Leu Ala Gly AlaIle

1 5 10

<210>101

<211>13

<212>PRT

<213>Japanese encephalitis virus

<400>101

Ser Gln Glu Gly Gly Leu His His Ala Leu Ala Gly Ala

1 5 10

<210>102

<211>12

<212>PRT

<213>Japanese encephalitis virus

<400>102

Ser Gln Glu Gly Gly Leu His His Ala Leu Ala Gly

1 5 10

<210>103

<211>11

<212>PRT

<213>Japanese encephalitis virus

<400>103

Ser Gln Glu Gly Gly Leu His His Ala Leu Ala

1 5 10

<210>104

<211>10

<212>PRT

<213>Japanese encephalitis virus

<400>104

Ser Gln Glu Gly Gly Leu His His Ala Leu

1 5 10

<210>105

<211>9

<212>PRT

<213>Japanese encephalitis virus

<400>105

Ser Gln Glu Gly Gly Leu His His Ala

1 5

<210>106

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>106

Ser Gln Glu Gly Gly Leu His His

1 5

<210>107

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>107

Gln Glu Gly Gly Leu His His Ala

1 5

<210>108

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>108

Glu Gly Gly Leu His His Ala

1 5

<210>109

<211>6

<212>PRT

<213>Japanese encephalitis virus

<400>109

Gly Gly Leu His His Ala

1 5

<210>110

<211>5

<212>PRT

<213>Japanese encephalitis virus

<400>110

Gly Leu His His Ala

1 5

<210>111

<211>4

<212>PRT

<213>Japanese encephalitis virus

<400>111

Leu His His Ala

1

<210>112

<211>15

<212>PRT

<213>Japanese encephalitis virus

<400>112

Thr Glu Lys Phe Ser Phe Ala Lys Asn Pro Val Asp Thr Gly His

1 5 10 15

<210>113

<211>14

<212>PRT

<213>Japanese encephalitis virus

<400>113

Thr Glu Lys Phe Ser Phe Ala Lys Asn Pro Val Asp Thr Gly

1 5 10

<210>114

<211>13

<212>PRT

<213>Japanese encephalitis virus

<400>114

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

1 5 10

<210>115

<211>12

<212>PRT

<213>Japanese encephalitis virus

<400>115

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

1 5 10

<210>116

<211>11

<212>PRT

<213>Japanese encephalitis virus

<400>116

Thr Glu Lys Phe Ser Phe Ala Lys Asn Pro Val

1 5 10

<210>117

<211>10

<212>PRT

<213>Japanese encephalitis virus

<400>117

Thr Glu Lys Phe Ser Phe Ala Lys Asn Pro

1 5 10

<210>118

<211>9

<212>PRT

<213>Japanese encephalitis virus

<400>118

Thr Glu Lys Phe Ser Phe Ala Lys Asn

15

<210>119

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>119

Thr Glu Lys Phe Ser Phe Ala Lys

1 5

<210>120

<211>11

<212>PRT

<213>Japanese encephalitis virus

<400>120

Glu Lys Phe Ser Phe Ala Lys Asn Pro Val Asp

1 5 10

<210>121

<211>10

<212>PRT

<213>Japanese encephalitis virus

<400>121

Lys Phe Ser Phe Ala Lys Asn Pro Val Asp

1 5 10

<210>122

<211>9

<212>PRT

<213>Japanese encephalitis virus

<400>122

Phe Ser Phe Ala Lys Asn Pro Val Asp

1 5

<210>123

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>123

Ser Phe Ala Lys Asn Pro Val Asp

1 5

<210>124

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>124

Phe Ala Lys Asn Pro Val Asp

1 5

<210>125

<211>6

<212>PRT

<213>Japanese encephalitis virus

<400>125

Ala Lys Asn Pro Val Asp

1 5

<210>126

<211>5

<212>PRT

<213>Japanese encephalitis virus

<400>126

Lys Asn Pro Val Asp

1 5

<210>127

<211>4

<212>PRT

<213>Japanese encephalitis virus

<400>127

Asn Pro Val Asp

1

<210>128

<211>12

<212>PRT

<213>Japanese encephalitis virus

<400>128

Val Thr Val Asn Pro Phe Val Ala Thr Ser Ser Ala

1 5 10

<210>129

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>129

Val Asn Pro Phe Val Ala Thr Ser

1 5

<210>130

<211>4

<212>PRT

<213>Japanese encephalitis virus

<400>130

Pro Phe Val Ala

1

<210>131

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>131

Pro Phe Gly Asp Ser Tyr Ile Val

1 5

<210>132

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>132

Val Gly Arg Gly Asp Lys Gln Ile

1 5

<210>133

<211>12

<212>PRT

<213>Japanese encephalitis virus

<400>133

Gly Asp Ser Tyr Ile Val Val Gly Arg Gly Asp Lys

1 5 10

<210>134

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>134

Ser Tyr Ile Val Val Gly Arg Gly

1 5

<210>135

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>135

Asn His His Trp His Lys Ala Gly

1 5

<210>136

<211>12

<212>PRT

<213>Japanese encephalitis virus

<400>136

Arg Gly Asp Lys Gln Ile Asn His His Trp His Lys

1 5 10

<210>137

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>137

Asp Lys Gln Ile Asn His His Trp

1 5

<210>138

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>138

Val Gly Arg Gly Asp Lys Gln

1 5

<210>139

<211>6

<212>PRT

<213>Japanese encephalitis virus

<400>139

Val Gly Arg Gly Asp Lys

1 5

<210>140

<211>5

<212>PRT

<213>Japanese encephalitis virus

<400>140

Val Gly Arg Gly Asp

1 5

<210>141

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>141

Gly Arg Gly Asp Lys Gln Ile

1 5

<210>142

<211>6

<212>PRT

<213>Japanese encephalitis virus

<400>142

Arg Gly Asp Lys Gln Ile

1 5

<210>143

<211>5

<212>PRT

<213>Japanese encephalitis virus

<400>143

Gly Asp Lys Gln Ile

1 5

<210>144

<211>8

<212>PRT

<213>Japanese encephalitis virus

<400>144

Gly Asn Arg Asp Phe Ile Gln Gly

1 5

<210>145

<211>8

<212>PRT

<213>Dengue virus type 1

<400>145

Gly Asn Arg Asp Phe Val Glu Gly

1 5

<210>146

<211>8

<212>PRT

<213>Murray Valley encephalitis virus

<400>146

Ser Ser Arg Asp Phe Leu Glu Gly

1 5

<210>147

<211>8

<212>PRT

<213>Murray Valley encephalitis virus

<400>147

Ser Ser Arg Asp Phe Ile Glu Gly

1 5

<210>148

<211>8

<212>PRT

<213>Kunjin virus

<400>148

Ser Asn Arg Asp Phe Leu Glu Gly

1 5

<210>149

<211>8

<212>PRT

<213>Tick-borne encephalitis virus

<400>149

Glu Asn Arg Asp Phe Val Thr Gly

1 5

<210>150

<211>8

<212>PRT

<213>Dengue virus type 2

<400>150

Ser Asn Arg Asp Phe Val Glu Gly

1 5

<210>151

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>151

Glu Asn His Gly Asn Tyr Thr

1 5

<210>152

<211>7

<212>PRT

<213>Murray Valley encephalitis virus

<400>152

Thr Asn His Gly Asp Tyr Ser

1 5

<210>153

<211>7

<212>PRT

<213>Murray Valley encephalitis virus

<400>153

Thr Ser His Gly Asn Tyr Ser

1 5

<210>154

<211>7

<212>PRT

<213>Kunjin virus

<400>154

Glu Ser His Gly Asn Tyr Phe

1 5

<210>155

<211>7

<212>PRT

<213>West Nile virus

<400>155

Glu Ser His Gly Asn Tyr Ser

1 5

<210>156

<211>7

<212>PRT

<213>Vsutu Virus

<400>156

Asp Thr His Gly Asn Tyr Ser

1 5

<210>157

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>157

Glu Gly Gly Leu His Gln Ala

1 5

<210>158

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>158

Glu Arg Ala Leu His Gln Ala

1 5

<210>159

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>159

Glu Gly Ser Leu His Gln Ala

1 5

<210>160

<211>7

<212>PRT

<213>Murray Valley encephalitis virus

<400>160

Glu Gly Ala Leu His Gln Ala

1 5

<210>161

<211>7

<212>PRT

<213>Tick-borne encephalitis virus

<400>161

Thr Gly Val Leu Leu Lys Ala

1 5

<210>162

<211>7

<212>PRT

<213>Dengue virus

<400>162

Glu Gly Ala Met His Thr Ala

1 5

<210>163

<211>7

<212>PRT

<213>Dengue virus type 4

<400>163

Glu Gly Ala Met His Ser Ala

1 5

<210>164

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>164

Phe Ala Lys Asn Pro Ala Asp

1 5

<210>165

<211>7

<212>PRT

<213>Japanese encephalitis virus

<400>165

Phe Arg Lys Asn Pro Ala Asp

1 5

<210>166

<211>7

<212>PRT

<213>Murray Valley encephalitis virus

<400>166

Phe Thr Lys Thr Pro Ala Asp

1 5

<210>167

<211>7

<212>PRT

<213>Murray Valley encephalitis virus

<400>167

Phe Ser Lys Asn Pro Ala Asp

1 5

<210>168

<211>7

<212>PRT

<213>Kunjin virus

<400>168

Phe Leu Gly Thr Pro Ala Asp

1 5

<210>169

<211>7

<212>PRT

<213>West Nile virus

<400>169

Phe Ala Arg Thr Pro Ala Asp

1 5

Claims (16)

1. an antigen epitope polypeptide is characterized in that, it contains any one aminoacid sequence shown in SEQ ID NO:64, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:108, SEQ ID NO:124, the SEQ ID NO:139.

2. antigen epitope polypeptide according to claim 1 is characterized in that the 7th amino acids Glu of the aminoacid sequence shown in the described SEQ ID NO:64 is replaceable and is Gln.

3. antigen epitope polypeptide according to claim 1 is characterized in that the 7th amino acids Ser of the aminoacid sequence shown in the described SEQ ID NO:95 is replaceable and is Thr.

4. antigen epitope polypeptide according to claim 1 is characterized in that the 6th amino acids His of the aminoacid sequence shown in the described SEQ ID NO:108 is replaceable and is Gln.

5. antigen epitope polypeptide according to claim 1 is characterized in that the 6th amino acids Val of the aminoacid sequence shown in the described SEQ ID NO:124 is replaceable and is Ala.

6. according to each described antigen epitope polypeptide of claim 1 to 5, it is characterized in that its aminoacid sequence is any one shown in SEQ ID NO:64, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:108, SEQID NO:124, the SEQ ID NO:139.

7, the nucleotide sequence of each described antigen epitope polypeptide of coding claim 1 to 5.

8, the nucleotide sequence of the described antigen epitope polypeptide of coding claim 6.

9, according to each described antigen epitope polypeptide of claim 1 to 5, it is characterized in that: described antigen epitope polypeptide carries out chemically modified at N end or C end.

10, antigen epitope polypeptide according to claim 6 is characterized in that: described antigen epitope polypeptide carries out chemically modified at N end or C end.

11, antigen epitope polypeptide according to claim 9 is characterized in that: the chemically modified of described polypeptide chain N end is the natural amination or the acetylize of N end; The chemical modification of described polypeptide chain C end is the carboxylated naturally or amidation of C end.

12, antigen epitope polypeptide according to claim 10 is characterized in that: the chemically modified of described polypeptide chain N end is the natural amination or the acetylize of N end; The chemical modification of described polypeptide chain C end is the carboxylated naturally or amidation of C end.

13, the vaccine composition of a kind of prevention or treatment encephalitis b virus is characterized in that: contain each described antigen epitope polypeptide of claim 1 to 5 that significant quantity is gone up in treatment.

14, the vaccine composition of a kind of prevention or treatment encephalitis b virus is characterized in that: contain the described antigen epitope polypeptide of claim 6 that significant quantity is gone up in treatment.

15, the purposes of each described antigen epitope polypeptide of claim 1 to 5 in preparation prevention or treatment encephalitis b virus medicine.

16, the purposes of the described antigen epitope polypeptide of claim 6 in preparation diagnosis or detection encephalitis b virus and antibody reagent thereof.

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