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CTL epitope polypeptide of avian influenza H5N1 virus and application thereof

A technology of epitope polypeptide and avian influenza, which is applied in the fields of application, antiviral agents, and medical preparations containing active ingredients, etc., can solve the problems that the H5N1 avian influenza virus T cell detection technology has not been established, and there is no H5N1 virus, etc.

Inactive Publication Date: 2009-10-21
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For avian influenza H5N1 virus, if its specific CTL epitopes different from other influenza virus subtypes are determined, the corresponding specific CTL epitopes can be qualitatively and quantitatively analyzed, but since there is no H5N1 virus-specific T cell epitope, T cell detection technology for H5N1 avian influenza virus has not been established so far

Method used

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  • CTL epitope polypeptide of avian influenza H5N1 virus and application thereof
  • CTL epitope polypeptide of avian influenza H5N1 virus and application thereof
  • CTL epitope polypeptide of avian influenza H5N1 virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Using refolding experiment to detect the binding ability of polypeptide and HLA-A*0201 molecule

[0048] In the refolding experiment, if the polypeptide can bind to the HLA-A*0201 molecule, then the polypeptide, HLA-A*0201 heavy chain and β2m can form a stable polypeptide-MHC-β2m ternary complex, which is shown in the molecular sieve chromatography A polypeptide-heavy chain-light chain complex peak appears.

[0049] 1. Preparation of HLA-A*0201 Heavy Chain Extracellular Region Inclusion Body

[0050] 1. Construction of recombinant plasmid I

[0051] 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.

[0052] 2. Preparation of HLA-A*0201 Heavy Chain Extracellular Region Inclusion Body

[0053] The monoclonal strain of Escherichia coli BL21 (NEB Company, Cat. No. C2566H) transfected with the recombinant pl...

Embodiment 2

[0063] Example 2. Using T2 cell binding assay to detect the binding ability of polypeptides to HLA-A2 molecules on the surface of T2 cells

[0064] 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.

[0065] 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 phase, wash them twice with se...

Embodiment 3

[0067] 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

[0068] HLA-A*0201 / K b Transgenic mice: purchased from Shanghai Second Military Medical University.

[0069] The preparation method of recombinant plasmid III (pcDNA3.1(+) inserted into Qinghai Lake H5N1 virus H5HA) is as follows:

[0070] Design the upstream primer (5'-GCTAGCCATGGAGAAAATAGTGCTT-3') and the downstream primer (5'-AATGCAAATTCTGCATTGTAAAAGCTT-3') to amplify the gene of H5HA (GENBANK ACCESSIONGNO.AAZ16277), cut with Nhe I and HindIII, and simultaneously digest pcDNA3.1 (+) The vector (Invitrogen, Catalog nos. V790-20) was double-digested with Nhe I and HindIII, and the digested product was placed at 18°C ​​for overnight ligation, and then transformed into Escherichia coli DH5α (TAKARA, Cat. No. D9057). After clone screening and sequen...

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Abstract

The invention discloses a CTL epitope polypeptide of an avian influenza H5N1 virus and application thereof. The CTL epitope polypeptide of the avian influenza H5N1 virus is as shown by (a) or (b), wherein (a) is a polypeptide consisting of amino acid sequences shown by a sequence 1 in a sequence table; and (b) is a polypeptide derived from the sequence 1, is obtained by substituting and / or deleting and / or adding one or more amino acid residues for the amino acid sequence of the sequence 1 and is correlative with a CTL epitope of the avian influenza H5N1 virus. The DNA for coding the polypeptide is protected in the invention. The epitope polypeptide can be applied to preparing vaccines aiming at the avian influenza H5N1 virus and also can be applied to preparing a kit for testing specific T cells of the avian influenza H5N1 virus. The CTL epitope polypeptide of the avian influenza H5N1 virus has great 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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