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B cell antigen epitope polypeptide of infectious bronchitis virus isolate S1 protein, and vaccine thereof

A technology of antigenic epitope and cell antigen, applied in the field of genetic engineering and protein engineering, can solve the problem of few reports of B cell antigenic epitope, and achieve the effect of inhibiting IBV detoxification

Active Publication Date: 2016-09-21
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, most studies on the B cell epitope of IBV mainly focus on the N protein and M protein, and there are relatively few reports on the B cell epitope of the S protein.

Method used

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  • B cell antigen epitope polypeptide of infectious bronchitis virus isolate S1 protein, and vaccine thereof
  • B cell antigen epitope polypeptide of infectious bronchitis virus isolate S1 protein, and vaccine thereof
  • B cell antigen epitope polypeptide of infectious bronchitis virus isolate S1 protein, and vaccine thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Using bioinformatics method to analyze IBV Shanghai 1208 strain S1 protein B cell epitope

[0047] The amino acid sequence of the S1 gene of IBV Shanghai 1208 strain is shown in Table 1 (SEQ ID NO.1), using the web server BepiPred ( http: / / www.cbs.dtu.dk / services / BepiPred / ) The B cell epitope prediction tool calculates the amino acid properties of the S1 protein (hydrophilicity, flexibility, accessibility, polarity, exposed surface and turning angle, etc.) to predict B cell linear epitopes. Hydrophobicity and hydrophilicity. In the body, hydrophobic residues are generally buried inside the protein, while hydrophilic residues are located on the surface of the protein. Therefore, the hydrophilic part of the protein is related to the protein epitope. There is a close relationship; accessibility, the possibility of amino acid residues in protein antigens being contacted by solvent molecules, reflects the distribution of various amino acid residues of protein antigen...

Embodiment 2E

[0054] Example 2 Screening of functional B cell antigen epitopes by ELISA method

[0055] Artificial synthesis of predicted IBV S1 epitope polypeptides, each polypeptide synthesis amount is 10mg, the polypeptide is coupled with BSA to enhance immunogenicity, and the purity of the synthesized polypeptide is greater than 90%. Each synthetic polypeptide is mixed with Freund’s complete adjuvant and Freund’s incomplete adjuvant at a ratio of 1:1 to immunize SPF chickens separately. The first exemption is to use Freund’s complete adjuvant mixed polypeptide to inject 150ug subcutaneously into the chicken. Synthetic peptides for booster and Freund’s incomplete adjuvant are boosted 21 days after the initial immunization. The injection site and dose are the same. Chicken wing venous blood is collected every 7 days during the immunization period, and the serum is separated for the detection of B cell epitope antibodies .

[0056] Purify the prokaryotic expression of IBV S1 protein and coat i...

Embodiment 3B

[0059] Example 3 Construction of B cell epitope cassette eukaryotic expression plasmid (pV-S1B) and S1 gene expression plasmid (pV-S1)

[0060] A flexible linker (amino acid sequence is GGGGSGGGGSGGGGS (SEQ ID NO. 10)) is added between the screened B cell epitope sequences to maintain the secondary structure between epitopes to be folded correctly, and the functional IBV S1 protein B is synthesized by artificial gene synthesis. The cell epitopes are connected in series to form a B cell epitope box, as shown in Table 4 (SEQ ID NO. 11), named S1B. The 5'end of the synthesized S1B epitope box is added with the restriction enzyme Hind III restriction site and the Kozak sequence (amino acid sequence is GCCACCATG (SEQ ID NO. 12)), and the 3'end is added with the restriction enzyme Xho I enzyme Cut site.

[0061] Table 4 S1B epitope box amino acid sequence

[0062] NYVYYYQSAFRPPNGWHLQGGAYAVVNSINKTNNAGA GGGGSGGGGSGGGGGS AFRPPN GWHL GGGGSGGGGSGGGGGS NKTNNAGAASE GGGGSGGGGSGGGGGS TNGNDVG...

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Abstract

The invention provides a B cell antigen epitope polypeptide of a chicken infectious bronchitis virus Shanghai 1208 isolate S1 protein, and belongs to the field of gene and protein engineering. The B cell antigen epitope polypeptide can be combined to prepare an IBV multi-epitope vaccine. The IBV B cell antigen epitope can cause high-level immunoresponse.

Description

Technical field [0001] The present invention belongs to the fields of genetic engineering and protein engineering. Specifically, the present invention relates to a functional B cell epitope polypeptide of the S1 protein of an infectious bronchitis virus isolate and a vaccine thereof. Background technique [0002] Avian infectious bronchitis (Avian Infectious Bronchitis, IB) is one of the poultry B / II infectious diseases regulated by the OIE and my country. The disease is an acute, highly infectious and economically significant viral disease caused by Infectious Bronchitis Virus (IBV). The virus disease occurs in all poultry-raising countries, and the pathogen has multiple clinical phenotypes, including renal type, respiratory type, and proventriculus type, and it often causes mixed infections with viruses such as Newcastle disease (NDV) and avian influenza (AIV) , Bring greater difficulties to the prevention and control of the disease. At present, all three subtypes of IBV have...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/215A61P31/14G01N33/68
CPCA61K39/12A61K2039/53A61K2039/552C07K14/005C07K2319/00C12N2770/20022C12N2770/20034G01N33/68G01N33/6854
Inventor 丁铲谭磊于圣青孙英杰仇旭升孟春春宋翠萍
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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