CN102276700B - Swine hepatitis E virus antigen epitope and application thereof - Google Patents
Swine hepatitis E virus antigen epitope and application thereof Download PDFInfo
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Abstract
The invention discloses a swine hepatitis E virus antigen epitope and applications thereof, and the sequence of the swine hepatitis E virus antigen epitope is shown in SEQ ID No:8. The swine hepatitis E virus antigen epitope obtained by computer software screening and experiment validation, on one hand, is applicable to rapid specific swine hepatitis E diagnostic kits, and can be used for rapid diagnosis of swine hepatitis E, and especially for the development of differential diagnosis technology of vaccine immune antibody and natural inapparent infection antibody for the monitoring of swine hepatitis E prevalence; on the other hand, can also be used to design specific, safe and high-efficient swine hepatitis E vaccines for the prevention of swine hepatitis E, the control of swine hepatitis E prevalence, and the obstruction of hepatitis E spreading from swine to human body.
Description
The application is to be on April 1st, 2009 applying date, and application number is 200910048702.6, and denomination of invention is divided an application for the application for a patent for invention of " swine hepatitis E virus epitope and application thereof ".
Technical field
The present invention relates to a kind of hepatitis virus antigen epi-position, relate in particular to a kind of swine hepatitis E virus epitope and the application in vitro detection and vaccine thereof.
Background technology
Hepatitis E virus (Hepatitis E virus, HEV) is a kind of non-first of propagating through excrement, mouthful approach-non-hepatitis b disease poison.This virus can cause outbreak of epidemic the crowd, and main infringement is between twenty and fifty, can cause 20% case fatality rate to the pregnant woman.1986-1988 Xinjiang of China southern areas cause hepatitis E popular because of contaminated drinking water, 119280 examples of falling ill altogether, and dead 707 examples are that a hepatitis E of maximum is popular in the world so far.The hepatitis E sickness rate has the trend that increases year by year in recent years, and according to China Ministry of Health statistics, viral hepatitis type E sickness rate ratio in 2003 rose 40.4% in 2002.Therefore, the work schedule of public health services at different levels has been listed in the viral hepatitis type E prevention and control in.
Recent study finds, the animals such as HEV can infected pigs, ox, sheep, mouse and chicken.Wherein, virus is higher than 50% in the antibody positive rate of pig and mouse, infers that thus animal is the important intermediate host that HEV propagates.Gene sequencing shows, the hepatitis E virus that separates in the animal body and people's HEV have high dna homolog, and the edible Wild boar as food of existing people and venison and infect the report of viral hepatitis type E, prove viral hepatitis type E be a kind of can be by food-borne infecting both domestic animals and human epidemic disease.(Chinese Amphixenosis's magazine such as Ge Shengxiang, 2003,19 (2), 108-109) the HEV serosurvey has been carried out in 8626 Adult Pigs on 120 pig farms of 20 provinces, municipalities and autonomous regions of China, the result shows that average infection positive rate is 83.4%, wherein the highest province is the Inner Mongol (100%), and minimum is Hubei (68%).In view of the high infection rate of China HEV, epi-position to pig viral hepatitis type E virus is studied, set up special, effective pig viral hepatitis type E detection method, develop safe and effective pig viral hepatitis type E vaccine, the popular occurrence regularity of understanding, block this disease by the propagation of pig to the people, for guaranteeing that the people's is healthy and safe significant.
The scholar Qi of domestic The 2nd Army Medical College (Journal of Virological Methods 1995,5,55-66) open reading frame ORF1, the ORF2 of people HEV supposition, the albumen order of ORF3 have been analyzed, 7 peptides have been synthesized, find that wherein 3 peptides have immunogenicity, can be used as the diagnosis of HEV.(Virology 2001,288,203-211) amplify the recombinant peptide section of the intermeshing of different sizes from people HEV ORF2, find that one of fragment wherein---pB166 is neutralizing epitope, can be used as vaccine development and the diagnosis of HEV for American scholar Meng.(Virology 2002,293, reported that 273-280) 11 amino acid peptide sections of C-terminal of people HEV-VLP have immunogenicity, can induce generation antibody for Japanese scholars Niikura.The scholar Zhang of Xiamen University (Vaccine 2005,23,2881-2892) finder HEV coat protein 394-606 position peptide section with dimeric form can with HEV human serum kickback, as vaccine can anti-macaque experimental infection.(Vaccine 2007,25, and 4350-4360) the whole person HEV ORF2 albumen of report expression and the inner 458-607 of ORF2 position peptide section have immunogenicity, play neutralizing effect for India scholar Deshmukh.Can be used as the exploitation of vaccine.But, up to the present, also do not have the screening system report of pig HEV open reading frame ORF1, albumen epi-position that ORF2 is relevant with ORF3.
Although the research of pig viral hepatitis type E more and more obtains global attention, the research of pig viral hepatitis type E is limited to more at present epidemiology survey and some simple zoogenetic infection tests of regional area both at home and abroad.Followingly remain to be answered a question and technical bottleneck still having aspect the pig viral hepatitis type E prevention and control research.Also do not have at present the epi-position research report of pig viral hepatitis type E virus, the test kit that is used for the detection of pig viral hepatitis type E on the market is the test kit that detects for people's viral hepatitis type E.Although pig viral hepatitis type E virus is very high with people's viral hepatitis type E virus sequence homology, but between them after all or some difference, the viral hepatitis type E virus epitopes of employment detects pig viral hepatitis type E virus and causes the specificity (Speciality) that detects not strong, can not specially reflect pig viral hepatitis type E regularty of epidemic.
Summary of the invention
One object of the present invention is to provide a kind of swine hepatitis E virus epitope.
Another object of the present invention is to provide a kind of polypeptide for the pig hepatitis E vitro detection.
Another purpose of the present invention is to provide a kind of polypeptide for the preparation of pig penta type liver vaccine.
The present invention utilizes computer software to screen the identification epi-position from the corresponding albumen of the full genome open reading frame of pig hepatitis E virus ORF1, ORF2 and ORF3.The Main Basis of epi-position identification is that the secondary structure of spiral, sheet and corner, Robson-Garnier of the wetting ability of protein or polypeptid acid sequence, surperficial degree, suppleness, Chou-Fasman and C-and N-end are predicted antigen integrated data (Antigenic Index, AI).Utilize existing software, as: GCG (Madison, Wisconsin, USA) and DNAStar (Madison, Wisconsin, USA) calculate AI and aid identification antigenic region from the albumen of ORF1, the ORF2 of pig HEV and ORF3.According to the aminoacid sequence that the known protein of ORF1, the ORF2 of pig HEV and ORF3 is obtained, estimate again their antigenic region (AI) (general 10 to 15 amino acid) with computer program.
A kind of swine hepatitis E virus epitope, its aminoacid sequence is as follows:
Val-Glu-His-Asn-Pro-Lys-Arg-Leu-Glu-Ala-Ala-Tyr-Arg-Glu-Thr-Cys-Ser-Arg-Arg-Gly-Thr-Ala-Ala-Tyr-Pro-Leu-Leu-Gly-Ala-Gly-Ile-Tyr-Lys-Val-Pro-Val-Gly-Leu-Ser-Phe-Asp-Ala-Trp-Glu-Arg-Asn-His-Arg-Pro-Gly-Asp-Glu。
Another kind of swine hepatitis E virus epitope, its aminoacid sequence is as follows:
Ser-Asp-Ser-Val-Leu-Thr-Phe-Glu-Leu-Thr-Asp-Ile-Val-His-Cys-Arg-Met-Ala-Ala-Pro-Ser-Gln-Arg-Lys-Ala-Val-Leu-Ser-Thr-Leu-Val-Gly-Arg-Tyr-Gly-Arg-Arg-Thr-Lys-Leu-Tyr-Glu-Ala-Ala-His-Ala-Asp-Val-Arg-Gly-Ser。
Another kind of swine hepatitis E virus epitope, its aminoacid sequence is as follows:
Pro-Arg-Gln-Pro-Ala-Arg-Pro-Leu-Gly-Ser-Ala-Trp-Arg-Asp-Gln-Ser-Gln-Arg-Pro-Ala-Ala-Ser-Thr-Arg-Arg-Arg-Pro-Ala-Pro-Ala-Gly-Ala-Ser-Pro-Leu-Thr-Ala-Val-Ala-Pro-Ala-Pro-Asp-Thr-Ala-Pro-Val-Pro-Asp-Val-Asp-Ser-Arg-Gly-Ala-Ile-Leu-Arg-Arg-Gln-Tyr-Asn-Leu-Ser。
Another kind of swine hepatitis E virus epitope, its aminoacid sequence is as follows:
Pro-Val-Ser-Ile-Ser-Ala-Val-Gly-Val-Leu-Ala-Pro-His-Ser-Ala-Leu-Ala-Ile-Leu-Glu-Asp-Thr-Ala-Asp-Tyr-Pro-Ala-Arg-Ala-His-Thr-Phe-Asp-Asp-Phe-Cys-Pro-Glu-Cys-Arg-Ser-Leu-Gly-Leu-Gln-Gly-Cys。
By analyzing, polypeptide Leu-Gly-Ala-Thr-Ser-Pro-Ser-Ala-Pro-Pro-Leu-Pro-Pro-Val-Val-Asp-Leu, polypeptide Pro-Pro-Val-Val-Asp-Leu-Pro-Gln-Leu-Gly, polypeptide Leu-Ala-Leu-Gly-Ser-Gln-Pro-Val-His-Ser-Ala-Pro-Leu-Gly-Ala-Thr-Ser-Pro-Ser-Ala-Pro-Pro-Leu, polypeptide Pro-Gln-Leu-Gly-Leu-Arg-Arg and polypeptide Leu-Ala-Leu-Gly-Ser-Gln-Pro-Val-His-Ser-Ala-Pro-Leu-Gly-Ala-Thr-Ser-Pro-Ser-Ala-Pro-Pro-Leu-Pro-Pro-Val-Val-Asp-Leu are the important component parts of swine hepatitis E virus epitope, it is the indispensable important component part of antibodies antigen recognition epi-position, these polypeptide can be independent, the N end or the C end that are connected or are connected in other polypeptide of the N of one peptide species end and another kind of peptide C end, described these polypeptide are independent or jointly the vitro detection of pig hepatitis E and the preparation of immune antibody are played an important role.
Another kind of swine hepatitis E virus epitope, its aminoacid sequence is as follows:
Ser-Pro-Ser-Pro-Ser-Pro-Ile-Phe-Ile-Gln-Pro-Thr-Pro-Ser-His-Leu-Thr-Phe-Gln-Pro-Gln-Pro-Gly-Leu-Glu-Leu-Ala-Leu-Gly-Ser-Gln-Pro-Val-His-Ser-Ala-Pro-Leu-Gly-Ala-Thr-Ser-Pro-Ser-Ala-Pro-Pro-Leu-Pro-Pro-Val-Val-Asp-Leu-Pro-Gln-Leu-Gly。
Another kind of swine hepatitis E virus epitope, its aminoacid sequence is as follows:
Leu-Ala-Leu-Gly-Ser-Gln-Pro-Val-His-Ser-Ala-Pro-Leu-Gly-Ala-Thr-Ser-Pro-Ser-Ala-Pro-Pro-Leu-Pro-Pro-Val-Val-Asp-Leu-Pro-Gln-Leu-Gly-Leu-Arg-Arg。
Another kind of swine hepatitis E virus epitope, its aminoacid sequence is as follows:
Leu-Gly-Ala-Thr-Ser-Pro-Ser-Ala-Pro-Pro-Leu-Pro-Pro-Val-Val-Asp-Leu-Pro-Gln-Leu-Gly-Leu-Arg-Arg。
Another kind of swine hepatitis E virus epitope, its aminoacid sequence is as follows:
Leu-Ala-Leu-Gly-Ser-Gln-Pro-Val-His-Ser-Ala-Pro-Leu-Gly-Ala-Thr-Ser-Pro-Ser-Ala-Pro-Pro-Leu。
Aforementioned polypeptides can be finished by the mode of chemosynthesis.The Fmoc method is adopted in chemosynthesis, and is synthetic by solid phase synthesis technique.The concrete steps of the method are referring to Eur.J.Immunol.1994,24,3188-3193; J.Org.Chem.1972,37,3404-3409; Huang Weide, old normal celebrating polypeptide is synthetic, Beijing: Science Press, 1985.
Aforementioned polypeptides can also be by forming fusion rotein antigen with other polypeptide or albumen, fusion rotein antigen can make by the genetically engineered mode, the i.e. engineering strain of transduceing/be converted into of DNA by the target fusion rotein, as: intestinal bacteria (E.coli), express and purifying in the yeast (Saccharomyces cerevisiae), or at Chinese hamster ovary (CHO) cell, the COS cell, little hamster kidney (BHK) cell, mouse myeloma SP2/O cell, express in the mammalian cells such as mouse NSO thymoma and purifying after, obtain wherein a kind of antigens c end, via amido linkage (amide bond) and the end to end fusion protein product of another kind of antigen N-terminal (Protein Expr.Purif., 2008,59,189-196).
Clone, expression and purifying that gene engineering method prepares proteantigen can be undertaken by several different methods.Concrete grammar is referring to " molecular cloning experiment guide " (third edition), Science Press, 2002, the 1217-1270 pages or leaves.Suitable prokaryotic expression carrier such as pEGX series prokaryotic expression carrier (Amersham Pharmacia), pET series prokaryotic expression carrier (Novagen) etc.PGEX-4T-2 carrier particularly preferably.
For the antigen that different peptide sections merge, well-known its methods and principles in this area.Particularly, need between different peptide sections, add a joint in order not affect the function separately for the treatment of the fusogenic peptide section.Selection about joint specifically can be referring to Protein Eng., 2001,14,529-532.
By aforementioned polypeptides alone or in combination or merge and antibody that the antigen that forms produces for detection of replying pig hepatitis E virus.Particularly, the application of these polypeptide in the pig hepatitis E vitro detection.
Detection method can have multiple, such as indirect enzyme-linked immunosorbent determination techniques, double antigens sandwich enzyme immunoassay technique, gold mark Fast Detection Technique, immunity percolation detection technique and protein chip detection technique etc.Indirect enzyme-linked immunosorbent is measured, and namely indirect ELISA is the most frequently used detection method.
The indirect ELISA method ultimate principle is: polypeptide (antigen) is coated on the enzyme plate surface, serum/plasma to be checked after the dilution and contrast are added in the reaction plate hole, if have antibody in the tested serum/plasma, behind incubation, then the antigenic peptide of specific antibody in reaction plate hole is combined in the serum/plasma, form solid phase antigen-antibody complex, other composition in the unconjugated serum/plasma of flush away, the anti-human IgG antibody that adds enzyme labelling, incubation, the antibody of solid phase antigen (polypeptide) combination again with the anti-human IgG antibodies of enzyme labelling, the unconjugated enzymic-labelled antibody composition of flush away, add enzyme substrates, and be catalyzed into and be coloured product, add at last the stop buffer termination reaction.According to contrast, but antagonist carries out quantitatively or qualitative test.
Aforementioned polypeptides alone or in combination or merge and the antigen that forms for the preparation of anti-pig hepatitis E virus antibody.
The beneficial effect that technical solution of the present invention realizes:
The present invention has obtained the epitope of pig viral hepatitis type E virus by computer software screening and experimental verification.It can be applied to alone or in combination quick special pig viral hepatitis type E diagnostic kit on the one hand, be used for the quick diagnosis of pig viral hepatitis type E, the differential diagnosis technology of the immune antibody that particularly develops vaccine and natural inapparent infection antibody is used for the popular monitoring of pig viral hepatitis type E; Also can design on the other hand pig viral hepatitis type E vaccine special, safety and efficiently is propagated to the people by pig for the prevention of pig viral hepatitis type E, the popular and blocking-up viral hepatitis type E of control pig viral hepatitis type E.
Embodiment
Below describe technical scheme of the present invention in detail, the embodiment of the invention is only unrestricted in order to technical scheme of the present invention to be described, although with reference to preferred embodiment the present invention is had been described in detail, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement the technical scheme of invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the claim scope of the present invention.
If the used reagent of the present invention does not clearly indicate, then all available from Sigma-aldrich (Sigma-Aldrich).
Embodiment 1 pig viral hepatitis type E viral surface antigen
Utilize computer software from the corresponding albumen of the full genome open reading frame of pig hepatitis E virus ORF1, ORF2 and ORF3, to screen the identification epi-position.The Main Basis of epi-position identification is that the secondary structure of spiral, sheet and corner, Robson-Garnier of the wetting ability of protein or polypeptid acid sequence, surperficial degree, suppleness, Chou-Fasman and C-and N-end are predicted antigen integrated data (Antigenic Index, AI).Utilize existing software, as: GCG (Madison, Wisconsin, USA) and DNAStar (Madison, Wisconsin, USA) calculate AI and aid identification antigenic region from the albumen of ORF1, the ORF2 of pig HEV and ORF3.According to the aminoacid sequence that the known protein of ORF1, the ORF2 of pig HEV and ORF3 is obtained, estimate again their antigenic region (AI) (general 10 to 15 amino acid) with computer program.
Advanced analysis, respectively from ORF1, ORF2 and the selected following polypeptide of ORF3:
ORF1：
swHEV-1：83-140
Ala-Gly-Arg-Cys-Leu-Glu-Val-Gly-Ala-His-Pro-Arg-Ser-Ile-Asn-Asp-Asn-Pro-Asn-Val-Leu-His-Arg-Cys-Phe-Leu-Lys-Pro-Val-Gly-Arg-Asp-Val-Gln-Arg-Trp-Tyr-Thr-Ala-Pro-Thr-Arg-Gly-Pro-Ala-Ala-Asn-Cys-Arg-Arg-Ser-Ala-Leu-Arg-Gly-Leu-Pro-Pro，
Its corresponding dna sequence dna is:
swHEV
N-1：281-454
ccgggcgctg?tcttgaggtg?ggtgcccacc?cacgttctat?taatgacaac?cctaacgtcctgcatcgctg?ctttcttaaa?cctgttggcc?gcgacgttca?gcggtggtat?accgctccta?cccgtggccctgcagcaaat?tgccggcggt?ccgctctccg?cgggcttcca?cctg。
swHEV-2：874-925
Val-Glu-His-Asn-Pro-Lys-Arg-Leu-Glu-Ala-Ala-Tyr-Arg-Glu-Thr-Cys-Ser-Arg-Arg-Gly-Thr-Ala-Ala-Tyr-Pro-Leu-Leu-Gly-Ala-Gly-Ile-Tyr-Lys-Val-Pro-Val-Gly-Leu-Ser-Phe-Asp-Ala-Trp-Glu-Arg-Asn-His-Arg-Pro-Gly-Asp-Glu，
Its corresponding dna sequence dna is:
swHEV
N-2：2654-2809
tcgaaca?taaccccaaa?cggctcgagg?ccgcctatcg?ggagacctgc?tcccgacgggggacggctgc?ttaccccctg?ctcggtgccg?gcatatataa?ggtccctgtt?gggttgagct?ttgatgcctgggagcgtaac?caccgacccg?gggatgaat。
swHEV-3：1295-1346
Ser-Asp-Ser-Val-Leu-Thr-Phe-Glu-Leu-Thr-Asp-Ile-Val-His-Cys-Arg-Met-Ala-Ala-Pro-Ser-Gln-Arg-Lys-Ala-Val-Leu-Ser-Thr-Leu-Val-Gly-Arg-Tyr-Gly-Arg-Arg-Thr-Lys-Leu-Tyr-Glu-Ala-Ala-His-Ala-Asp-Val-Arg-Gly-Ser，
Its corresponding dna sequence dna is:
swHEV
N-3：3917-4072
atag?tgtgctcact?tttgagctca?cggacatagt?gcactgccgt?atggcagcgc?ccagccagcgcaaggcggtc?ctgtcaactc?ttgtcggcag?gtacggccga?cgtactaaat?tgtacgaggc?cgcacacgcagatgtccgcg?gatctctgaa?tc。
swHEV-4：1521-1582
Glu-Ser-Leu-Arg-Gly-Phe-Trp-Lys-Lys-His-Ser-Gly-Glu-Pro-Gly-Thr-Leu-Leu-Trp-Asn-Thr-Val-Trp-Asn-Met-Ala-Val-Ile-Ala-His-Cys-Tyr-Glu-Phe-Arg-Asp-Leu-Lys-Val-Ala-Ala-Phe-Lys-Gly-Asp-Asp-Ser-Val-Val-Leu-Cys-Ser-Asp-Tyr-Arg-Gln-Ser-Arg-Asp-Ala-Ala-Ala，
Its corresponding dna sequence dna is:
swHEV
N-4：4561-4746
gttcgctctg?cttgggttct?acaggcaccg?aaggagtctc?tgcgaggctt?ctggaagaaacactctggtg?agcctggcac?cctattatgg?aataccgttt?ggaacatggc?tgtcatagca?cactgttatgaattccgcga?ccttaaggtt?gcagcattca?agggggacga?ttctgttgtg?ctttgc。
ORF2：
swHEV-5：90-153
Pro-Arg-Gln-Pro-Ala-Arg-Pro-Leu-Gly-Ser-Ala-Trp-Arg-Asp-Gln-Ser-Gln-Arg-Pro-Ala-Ala-Ser-Thr-Arg-Arg-Arg-Pro-Ala-Pro-Ala-Gly-Ala-Ser-Pro-Leu-Thr-Ala-Val-Ala-Pro-Ala-Pro-Asp-Thr-Ala-Pro-Val-Pro-Asp-Val-Asp-Ser-Arg-Gly-Ala-Ile-Leu-Arg-Arg-Gln-Tyr-Asn-Leu-Ser，
Its corresponding dna sequence dna is:
swHEV
N-5：5422-5607
ctcggcagc?cagcccgtcc?actcggctcc?gcttggcgcg?accagtccca?gcgccccgccgcttccaccc?gtcgtcgacc?tgccccagct?ggggcttcgc?cgttgacggc?tgtggctccg?gctcctgacactgcacccgt?ccctgatgtt?gattcccgtg?gcgctatctt?gcgccgccag?tataatt
swHEV-6：433-476
Ala-Gln-Gln-Asp-Lys-Gly-Ile-Ala-Ile-Pro-His-Asp-Ile-Asp-Leu-Gly-Glu-Ser-Arg-Val-Val-Ile-Gln-Asp-Tyr-Asp-Asn-Gln-His-Glu-Gln-Asp-Arg-Pro-Thr-Pro-Ser-Pro-Ala-Pro-Ser-Arg-Pro-Phe，
Its corresponding dna sequence dna is:
swHEV
N-6：6451-6582
ctcagcagga?caagggtata?gctattccac?atgatattga?tctcggtgag?tcccgtgtggtcattcagga?ttatgataat?caacatgagc?aagaccgccc?caccccctct?cctgctccct?ctcgcccttt?tt。
swHEV-7：606-653
Pro-Val-Ser-Ile-Ser-Ala-Val-Gly-Val-Leu-Ala-Pro-His-Ser-Ala-Leu-Ala-Ile-Leu-Glu-Asp-Thr-Ala-Asp-Tyr-Pro-Ala-Arg-Ala-His-Thr-Phe-Asp-Asp-Phe-Cys-Pro-Glu-Cys-Arg-Ser-Leu-Gly-Leu-Gln-Gly-Cys，
Its corresponding dna sequence dna is:
swHEV
N-7：6970-7113
c?cgtgtctatt?tctgctgttg?gtgtccttgc?ccctcattct?gcgctggcta?ttctggaggacaccgctgat?taccctgctc?gcgcccatac?ttttgatgat?ttctgccctg?agtgccgttc?actcggccttcagggttgtg?ctt。
ORF3：
swHEV-8：22-58
Cys-Pro-Arg-His-Arg-Pro-Val-Ser-Pro-Leu-Ala-Val-Ala-Ala-Gly-Gly-Ala-Ala-Ala-Val-Pro-Ala-Val-Val-Ser-Gly-Val-Thr-Gly-Leu-Ile-Leu-Ser-Pro-Ser-Pro-Ser，
Its corresponding dna sequence dna is:
swHEV
N-8：5246-5356
gcccg?cgccaccggc?cggtcagccc?tctggccgtc?gccgcgggcg?gcgcagcggcggtgccggca?gtggtttctg?gggtgaccgg?gttgattctc?agcccttcgc?cctccc。
swHEV-9：55-112
Ser-Pro-Ser-Pro-Ser-Pro-Ile-Phe-Ile-Gln-Pro-Thr-Pro-Ser-His-Leu-Thr-Phe-Gln-Pro-Gln-Pro-Gly-Leu-Glu-Leu-Ala-Leu-Gly-Ser-Gln-Pro-Val-His-Ser-Ala-Pro-Leu-Gly-Ala-Thr-Ser-Pro-Ser-Ala-Pro-Pro-Leu-Pro-Pro-Val-Val-Asp-Leu-Pro-Gln-Leu-Gly，
Its corresponding dna sequence dna is:
swHEV
N-9：5345-5518
cttcgc?cctcccctat?attcatccaa?ccaacccctt?cgcatctgac?attccagccg?cagccggggctggagctcgc?cctcggcagc?cagcccgtcc?actcggctcc?gcttggcgcg?accagtcccagcgccccgcc?gcttccaccc?gtcgtcgacc?tgccccagct?ggggcttc。
swHEV-10：79-114
Leu-Ala-Leu-Gly-Ser-Gln-Pro-Val-His-Ser-Ala-Pro-Leu-Gly-Ala-Thr-Ser-Pro-Ser-Ala-Pro-Pro-Leu-Pro-Pro-Val-Val-Asp-Leu-Pro-Gln-Leu-Gly-Leu-Arg-Arg，
Its corresponding dna sequence dna is:
swHEV
N-10：5417-5524
tcgc?cctcggcagc?cagcccgtcc?actcggctcc?gcttggcgcg?accagtccca?gcgccccgccgcttccaccc?gtcgtcgacc?tgccccagct?ggggcttcgc?cgtt。
swHEV-11：91-114
Leu-Gly-Ala-Thr-Ser-Pro-Ser-Ala-Pro-Pro-Leu-Pro-Pro-Val-Val-Asp-Leu-Pro-Gln-Leu-Gly-Leu-Arg-Arg，
Its corresponding dna sequence dna is:
swHEV
N-11：5453-5524
ttggcgcg?accagtccca?gcgccccgcc?gcttccaccc?gtcgtcgacc?tgccccagctggggcttcgc?cgtt。
swHEV-12：79-101
Leu-Ala-Leu-Gly-Ser-Gln-Pro-Val-His-Ser-Ala-Pro-Leu-Gly-Ala-Thr-Ser-Pro-Ser-Ala-Pro-Pro-Leu，
Its corresponding dna sequence dna is:
swHEV
N-12：5417-5485
tcgc?cctcggcagc?cagcccgtcc?actcggctcc?gcttggcgcg?accagtccca?gcgccccgccgcttc。
After above-mentioned sequence is synthetic by Peptide synthesizer by GL-Biochem (shanghai) Ltd, obtain purity greater than 95% polypeptide by the HPLC purifying again.
The screening of embodiment 2 pig hepatitis E virus epi-positions
The total antibody ELISA test kit of pig HEV of producing with the safe industry biological medicine in Beijing ten thousand company limited is first identified the porcine blood serum that collects, and filters out pig HEV positive serum and negative serum.
The pig HEV polypeptide that with pig HEV positive serum and negative serum embodiment 1 is obtained again carries out ELISA to be identified, filters out pig HEV epitope antigen.Its concrete steps are as follows:
1. antigen diluent
Get the polypeptide that 10 μ l concentration are 10mg/ml (polypeptide that 5mg is synthetic is dissolved in 0.5ml dimethyl sulfoxide (DMSO) (DMSO) and gets) and 90ul PBS liquid mixing, the antigenic dilution solubility that obtains is 1 μ g/ μ l.Get 15 μ l antigenic dilutions and 5ml coating buffer mixing, the antigen diluent solubility that obtains is 3 μ g/ml again.Add antigen diluent solubility 100 μ l/ holes, 4 ℃ of coated 12h at enzyme plate.
2. sealing
Suck coated night, every hole adds 200 μ l confining liquids in 37 ℃ of sealing 2h.Confining liquid is 3% bovine serum albumin (BSA)/TBS liquid.
3. add primary antibodie
Wash plate 3 times with PBST liquid, every hole adds 100 μ l 1%BSA/TBST liquid and 10 μ l serum, and the concussion mixing is put 37 ℃ of incubation 1h.
4. adding two resists
Wash plate 5 times with PBST liquid, every hole adds the goat-anti pig IgG of 100 μ l horseradish peroxidase (HRP) marks or IgM (all available from Immunology Consultants Laboratory, Inc., USA) diluent, puts 37 ℃ of incubation 1h.Two is anti-with the dilution of 1%BSA/TBST liquid.
5. colour developing
Wash plate 5 times with PBST liquid, every hole adds substrate solution 100ul, 37 degree colour developing 10min.Substrate solution is by the 10ml citrate buffer solution, 0.4mlTMB and 33 μ l 3%H
2O
2Configuration forms.
6. stop
The stop buffer that every hole adds 50 μ l 1mol/l HCl comes termination reaction, surveys the OD value with microplate reader.When (sample OD value/negative control OD value)＞2.1 positive, when (sample OD value/negative control OD value)＜2.1 negative.Experimental result sees Table one, table two and table three.
Described reagent is as follows:
PBS liquid (500ml, pH7.4): 0.575g Na
2HPO
4With 0.114g NaH
2PO
4The adding distil water dissolving is settled to into 500ml.
Coating buffer (1000ml, pH9.6) (): 1.59g Na
2CO
3With 2.93g NaHCO
3The adding distil water dissolving is settled to into 1000ml.
TBS liquid (2000ml, pH8.6): 16g NaCl, 0.4g KCl and the dissolving of 2.42g Trizma-Base adding distil water are settled to into 2000ml.
TBST liquid (1000ml, the concentration of Tween-20 is 0.1%): 1000ml TBS liquid adds 1.0ml Tween-20.
Citrate buffer solution (100ml, pH3.5-4.0): 0.84g citric acid and the dissolving of 0.294g Trisodium Citrate adding distil water are settled to into 100ml.
Stop buffer (500ml, 1M HCl): the 40.88ml concentrated hydrochloric acid is dissolved in 459.12ml distilled water.
The immune response (for IgG) of the synthetic pig HEV polypeptide of table one and the liquidation of pig HEV positive blood
"+" expression is positive; "-" expression is negative.
The immune response (for IgG) of the synthetic pig HEV polypeptide of table two and pig HEV negative serum dish
"+" expression is positive; "-" expression is negative.
The immune response (for IgM) of the synthetic pig HEV polypeptide of table three and the liquidation of pig HEV positive blood
Through identifying that polypeptide swHEV-2, swHEV-3, swHEV-5, swHEV-7, swHEV-9, swHEV-10, swHEV-11, swHEV-12 are positive epi-positions, it is pig HEV epi-position.
Claims (2)
1. swine hepatitis E virus epitope, its sequence is shown in SEQ ID No 8.
2. the application of swine hepatitis E virus epitope claimed in claim 1 in the preparation of pig hepatitis E vaccine.
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