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CTL epitope polypeptides of bird flu H5N1 virus and applications thereof

An epitope polypeptide, avian influenza technology, applied in the application, antiviral agent, medical preparations containing active ingredients, etc., can solve the problem of no H5N1 virus, H5N1 avian influenza virus T cell detection technology has not been established and other problems

Inactive Publication Date: 2009-10-28
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because there is no H5N1 virus-specific T cell epitope, the T cell detection technology for H5N1 avian influenza virus has not been established so far.

Method used

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  • CTL epitope polypeptides of bird flu H5N1 virus and applications thereof
  • CTL epitope polypeptides of bird flu H5N1 virus and applications thereof
  • CTL epitope polypeptides of bird flu H5N1 virus and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Using refolding experiment to detect the binding ability of polypeptide and HLA-A*0201 molecule

[0052] In the refolding experiment, if the peptide can bind to the HLA-A*0201 molecule, then the peptide, HLA-A*0201 and β2m can form a stable peptide-MHC-β2m ternary complex, and the peptide appears in the molecular sieve chromatogram - Heavy chain-light chain complex peak.

[0053] 1. Preparation of HLA-A*0201 Heavy Chain Extracellular Region Inclusion Body

[0054] 1. Construction of recombinant plasmid I

[0055] The DNA encoding the extracellular region of the heavy chain of HLA-A*0201 (GENBANK ACCESSION NO. AY365426 from the 5' end nucleotides 73-897) was introduced into pET-28(a) to obtain recombinant plasmid I.

[0056] 2. Preparation of HLA-A*0201 Heavy Chain Extracellular Region Inclusion Body

[0057] The monoclonal strain of Escherichia coli BL21 (NEB Company, Cat. No. C2566H) transfected with the recombinant plasmid I was inoculated in a small amo...

Embodiment 2

[0071] Example 2. Using T2 cell binding assay to detect the binding ability of polypeptides to HLA-A2 molecules on the surface of T2 cells

[0072] T2 cells are HLA-A*0201 positive and TAP molecule deficient cell lines. The cells cannot process endogenous antigens, but can present exogenous antigenic peptides. In the absence of antigenic peptides, the expression of HLA-A2 molecules on the cell surface is at a low level. Once a suitable exogenous antigenic peptide binds to HLA-A2 molecules on the surface of T2 cells, the expression of HLA-A2 on the cell surface Strength will be greatly improved.

[0073] 1. Polypeptide RI-10

[0074] T2 cells (ATCC, Cat.No.CRL-1992) were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS, Gibico) (supplemented with 5.9% HEPES, 0.307% glutamic acid, 0.11% sodium pyruvate, 2 % sodium bicarbonate, 50u / ml penicillin, 50u / ml streptomycin), the incubator temperature is 37 ℃, containing 5% CO 2 . Take the cells in the logarithmic growth...

Embodiment 3

[0079] Example 3, ELISPOT analysis was used to detect the polypeptide-induced HLA-A*0201 / K immunized with the pcDNA3.1(+) vector carrying the H5HA gene b Ability of Splenocytes of Transgenic Mice to Produce Specific CTL Responses

[0080] HLA-A*0201 / K b Transgenic mice: purchased from Shanghai Second Military Medical University.

[0081] Preparation of recombinant plasmid III (pcDNA3.1(+) vector inserted into Qinghai Lake H5N1 virus H5HA): design upstream primer (5'-GCTAGCCATGGAGAAAATAGTGCTT-3') and downstream primer (5'-AATGCAAATTCTGCATTGTAAAAGCTT-3') to amplify H5HA Gene (GENBANK ACCESSIONGNO.AAZ16277) was digested with Nhe I and HindIII, and the pcDNA3.1(+) vector (Invitrogen, Catalog nos. V790-20) was digested with Nhe I and HindIII at the same time, and the digested product was placed in After ligation overnight at 18°C, Escherichia coli DH5α (TAKARA, Cat. No. D9057) was transformed. After clone screening and sequencing identification, it was ready for use.

[0082] 1...

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Abstract

The invention discloses a CTL epitope polypeptide of bird flu H5N1 virus and applications thereof. The CTL epitope polypeptide of bird flu H5N1 virus is shown as (a) or (b); (a) the polypeptide consists of amino acid sequences in the sequence I out of a sequence list; and (b) the polypeptide derives from the sequence 1 by the replacing and / or missing and / or adding of one or more amino acid residues of the amino acid sequence in the sequence 1 and is correlate to the CTL epitope of the bird flu H5N1 virus. The invention also protects the DNA that codes the polypeptide. The epitope polypeptide can be used for preparing the bacterin for the bird flu H5N1 virus and can be also used for preparing the kit for detecting the specific T cell of the H5N1 virus. The CTL epitope polypeptides and the applications thereof have important values.

Description

technical field [0001] The invention relates to a CTL epitope polypeptide of avian influenza H5N1 virus and application thereof. Background technique [0002] Human avian influenza H5N1 virus is a subtype of influenza virus with high pathogenicity and high lethality derived from poultry. In 1997, the first outbreak of human avian influenza infection occurred in Hong Kong, 18 people were infected, and 6 of them died. By 2003, human bird flu cases had reappeared worldwide. From the end of 2003 to the present, there have been 408 infections, including 205 deaths. Human avian influenza is generally transmitted from birds to humans, and direct infection between humans is only an occasional phenomenon. However, once the virus mutates or rearranges with the human influenza virus gene, it may produce a new strain adapted to human replication and direct human-to-human transmission, leading to an influenza pandemic. Although no sustained human-to-human transmission of the H5N1 vir...

Claims

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Application Information

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IPC IPC(8): C07K7/06C12N15/44C12N15/63C12N5/10C12N1/00A61K39/145A61K48/00A61P31/16C12Q1/68G01N33/68
Inventor 高福孙业平
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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