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Influenza A virus PB1 protein T cell epitope polypeptide fragment and application thereof

A technology of influenza vaccine and medicinal salt, which is applied in the direction of viral peptides, viruses, antiviral agents, etc., can solve the problems of incompletely clear synergistic mechanism, inability to resist virus attack, etc., and achieve the effect of stimulating virus-specific T cell immune response

Active Publication Date: 2021-10-22
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other studies have shown that certain epitopes can induce strong activity, but cannot resist virus attack
Therefore, although epitope-based vaccines have many advantages, the mechanism and strength of epitope function, the restriction of epitope, and the synergy mechanism between epitopes are still not completely clear, and even some The identified epitopes themselves are still controversial

Method used

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  • Influenza A virus PB1 protein T cell epitope polypeptide fragment and application thereof
  • Influenza A virus PB1 protein T cell epitope polypeptide fragment and application thereof
  • Influenza A virus PB1 protein T cell epitope polypeptide fragment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, preparation of nonapeptide derived from PB1

[0056] The amino acid sequence of influenza A virus (PR8) PB1 is shown in SEQ ID NO.1, which is used as the sequence source of the synthetic polypeptide fragment PB1 81-89. Artificially synthesized polypeptide fragments PB1 81-89, randomly constructed point mutations (corresponding mutant polypeptide fragments a, b) except the anchor amino acids of PB1 81-89, and the negative control hepatitis B virus HBc82-90 peptide used in the experiment (abbreviated as polypeptide fragment HBc82 -90).

[0057] The amino acid sequences of these four polypeptides are as follows (from left to right from the nitrogen terminal to the carbon terminal direction):

[0058] Polypeptide fragment PB1 81-89 (SEQ ID NO.2): GYAQTDCVL

[0059] Polypeptide fragment HBc82-90 (SEQ ID NO.3): RELVVSYVN

[0060] Mutant polypeptide fragment a (SEQ ID NO.10): GYAQADCVL

[0061] Mutant polypeptide fragment b (SEQ ID NO.11): GYAQTDAVL

[0062]...

Embodiment 2

[0063] Example 2, PB1 81-89 polypeptide fragment and H-2K D Molecular binding force

[0064] 1. In vitro refolding (refolding) test to detect peptide and H-2K D molecular binding capacity

[0065] In the in vitro refolding experiment, if the polypeptide can bind with H-2K D Molecular binding, then peptide, H-2K D And β2 microglobulin (β2m) can form a stable polypeptide-H-2K D -β2m ternary complex, polypeptide-H-2K appears in the molecular sieve chromatogram D - β2m complex protein peak. H-2K D The molecule is composed of a heavy chain and a light chain, the coding sequence of the heavy chain is shown in SEQ ID NO.4, and its amino acid sequence is shown in SEQ ID NO.5; the coding sequence of the light chain is shown in SEQ ID NO.6, and its The amino acid sequence is shown in SEQ ID NO.7.

[0066] (1) H-2K D Construction of heavy chain extracellular region expression plasmid

[0067]will code H-2K D The DNA of the extracellular region of the heavy chain (Genbank No. K...

Embodiment 3

[0084] Example 3, Insect Baculovirus Expression System Expressing Heat Shock Protein gp96

[0085] 1. pFastBac TM 1- Construction of gp96 plasmid

[0086] 1. Design and synthesis of gp96 primers: according to the sequence of the human gp96 gene in GenBank (the sequence number is NM_003299, its gene sequence is shown in SEQ ID NO.8, and its encoded amino acid sequence is shown in SEQ ID NO.9) as a template , an EcoRI restriction site was added to the 5' end of the gp96 gene, and an XbaI restriction site was added to the 3' end. The forward primer sequence is: 5'-G GAATTC ATGGGCAGCAGCCATCAT-3' (the underline is the EcoRI restriction site); the reverse primer sequence is: 5'-GC TCTAGA CTATTACAATTCATCTTTTTC-3' (the underline is the XbaI restriction site), commissioned Shanghai Sangon Bioengineering Technology Service Company to synthesize the primer, and sequenced to verify that the sequence of the primer was correct.

[0087] 2. Extract the mRNA of human liver cancer cell H...

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Abstract

The invention discloses an influenza A virus PB1 protein T cell epitope polypeptide fragment and application thereof. The invention firstly provides an influenza A virus PB1 protein T cell epitope polypeptide of which the amino acid sequence is SEQ ID No.2 or SEQ ID No.10 or SEQ ID No.11. The invention further provides application of the polypeptide in preparation of influenza vaccines. The influenza A virus PB1 protein T cell epitope polypeptide fragment is screened and identified, when the antigen epitope is inoculated, the gp96 protein is used as an immunologic adjuvant, the virus specific T cell immunoreaction is greatly stimulated, cross immune protection aiming at different subtypes of influenza virus strains can be effectively caused, and a foundation is laid for research and development of novel influenza virus vaccines with a cross protection function in the future.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to an influenza A virus PB1 protein T cell epitope polypeptide fragment and an application thereof. Background technique [0002] Influenza virus spreads rapidly, spreads widely, and is highly pathogenic. It is one of the most serious public health problems in the world today. In the 1930s, humans began to recognize influenza viruses and develop vaccines. However, influenza viruses are still important pathogens that threaten human and animal health today. Influenza viruses can be divided into several different subtypes according to their surface antigens of hemagglutinin (HA) and neuraminidase (neuraminidase, NA). To date, a total of 18 HA subtypes (H1 to H18) and 11 NA subtypes (N1 to N11) have been identified. During the process of infecting different hosts, influenza viruses mutate mainly in two aspects: reassortment of genome segments and amino acid variation of v...

Claims

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Application Information

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IPC IPC(8): C07K14/11A61K39/145A61K39/39A61P31/16
CPCC07K14/005A61K39/12A61K39/39A61P31/16C12N2760/16122C12N2760/16134A61K2039/55516Y02P20/55
Inventor 孟颂东王子豪张含郑华国鞠莹
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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