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CD8+T cell epitope polypeptide of S1 protein of chicken IBV (Infectious Bronchitis Virus) S1 protein

A technology of cell antigen and epitope polypeptide, applied in the fields of genetic engineering and protein engineering

Active Publication Date: 2013-11-13
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

At present, there are few studies on the T cell epitope of IBV, which has caused a great impact on the development of IBV universal epitope vaccine research. Therefore, the present invention focuses on screening the CD8+ T cell epitope of the IBV S1 protein

Method used

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  • CD8+T cell epitope polypeptide of S1 protein of chicken IBV (Infectious Bronchitis Virus) S1 protein
  • CD8+T cell epitope polypeptide of S1 protein of chicken IBV (Infectious Bronchitis Virus) S1 protein
  • CD8+T cell epitope polypeptide of S1 protein of chicken IBV (Infectious Bronchitis Virus) S1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0045] Cloning of embodiment 2 chicken infectious bronchitis virus S1 gene, construction of nucleic acid vaccine

[0046] Construct the IBV S1 protein eukaryotic expression plasmid and verify its immunogenicity according to the following steps:

[0047] (1) Amplification of multiple subtypes of IBV S1 gene

[0048]According to the nucleotide sequences of S1 gene of IBV M41 strain (DQ834384.1), T strain (AY775779.1), and QX sample strain (JQ088078.1) published by GenBank, synthetic primers were designed as shown in Table 2.

[0049] Table 2 is used to amplify the primer of S1 gene

[0050]

[0051] The above three pairs of primers were subjected to PCR amplification respectively. The templates are respectively the genomic DNA of chicken infectious bronchitis virus M41 strain, Australia T strain, and QX-like IBV strain ck / CH / TS / 201012.

[0052] The PCR reaction program was: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, exten...

Embodiment 3

[0058] Example 3 ELIspot method to detect and predict the secretion of IFN-γ in chicken spleen lymphocytes stimulated by polypeptides

[0059] Immunization of eukaryotic expression plasmids is equivalent to immune DNA vaccines. The main feature of DNA vaccines is that they can generate relatively strong cellular immune responses, that is, T lymphocyte immune responses. After two immunizations, some T cells will differentiate into specific immune antigens. The characteristic of this part of cells is that when stimulated by the same antigen, they will secrete a large amount of cytokines (such as IFN-γ) to activate naive T lymphocytes and transform them into killer T lymphocytes. Cells infected with viral antigens are killed. Therefore, the present invention isolates immunized chicken spleen lymphocytes and stimulates them with the polypeptides screened in Example 1 as viral antigen peptides. If the spleen lymphocytes secrete a large amount of cytokines (such as IFN-γ), it means ...

Embodiment 4

[0079] Example 4 Application of Chicken IBV S1 Protein CD8+T Cell Functional Antigen Epitope in Preparation of IBV Universal Vaccine

[0080] Utilize the 4 universal chicken infectious bronchitis virus S1 protein CD8+ T cell epitopes obtained in Example 3, add "KAA" (nucleotide sequence AAAGCTGCT) and "AAY" (nucleotide sequence GCCGCATAC), "GAAA" (nucleotide sequence GGCGCAGCAGCC), "KAAA" (nucleotide sequence AAAGCAGCCGCA) and other linkers to connect to construct the expression cassette, Afl II and Xho I restriction sites are added before and after the expression cassette, and the front end enzyme of the expression cassette A KOZAK sequence (the nucleotide sequence is GCCACCATG) was added after the cutting site to enhance the translation efficiency of the eukaryotic gene, and it was reduced to a nucleotide sequence through codon degeneracy, and Shanghai Sangon Biotechnology Co., Ltd. was entrusted to artificially synthesize this The expression cassette sequence is cloned into...

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Abstract

The invention provides universal CD8+T cell epitope polypeptide of a chicken IBV (Infectious Bronchitis Virus) S1 protein, and belongs to the field of gene and protein engineering. The epitope polypeptides are prepared by the following steps: screening 21 epitope polypeptides in accordance with binding motif sequences in amino acid sequences of the S1 genes of the IBV virus according to the binding motif sequences of haplotype chicken major histocompatibility complex (MHC) I-type molecules; then, taking lymphocytes of three constructed SPF (Specific Pathogen Free) chicken immunized by DNA (Deoxyribonucleic Acid) recombinant plasmids of the S1 genes containing different subtype IBVs, determining the capacity of the 21 polypeptides which induce chicken splenic lymphocytes to secrete interferon-gamma by using an ELIspot (Enzyme-Linked Immunospot Assay) method, and finally, screening to obtain four universal functional T cell epitope polypeptides of IBV, wherein the sequences are respectively shown in SEQ ID NO. 1-4. The four epitope polypeptides provided by the invention are combined in use to prepare a universal vaccine for IBV. The invention further provides a method for screening the epitope of the functional T cell of the S1 protein of the IBV.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and protein engineering, and in particular relates to a CD8+ T cell antigen epitope polypeptide of chicken IBV S1 protein. Background technique [0002] Chicken infectious bronchitis (Avian Infectious Bronchitis, IB) is one of the poultry B / II infectious diseases stipulated by the International Office of Epizootics (OIE) and my country. The disease is an acute, highly contagious and economically significant viral disease caused by Infectious Bronchitis Virus (IBV). Occurs in all poultry-raising countries, and the pathogen has multiple clinical phenotypes, including kidney type, respiratory type, and glandular stomach type, etc., and often causes mixed infections with Newcastle disease (AIV), avian influenza (AIV) and other viruses, giving the disease Disease control poses great difficulties. At present, all three subtypes of IBV have been found in chicken farms in my country. [0003] The IB...

Claims

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Application Information

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IPC IPC(8): C07K7/06G01N33/68A61K39/215A61P31/14C12N15/50
Inventor 丁铲谭磊仇旭升宋翠萍孙英杰于圣青范瑾
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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