Gene for coding recombinant avian influenza virus HA protein, virus-like particle, vaccine, preparation and application

A technology of avian influenza virus and bacmid, applied in the direction of recombinant DNA technology, application, virus, etc., can solve the problems of insufficient supply of chicken embryos, pollution, endogenous waste, etc., and achieve good cross protection and great optimization The effect of space, complete clinical protection

Active Publication Date: 2021-12-31
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, domestic bird flu vaccines are mainly inactivated whole virus vaccines, which rely on chicken embryos for production, and there a

Method used

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  • Gene for coding recombinant avian influenza virus HA protein, virus-like particle, vaccine, preparation and application
  • Gene for coding recombinant avian influenza virus HA protein, virus-like particle, vaccine, preparation and application
  • Gene for coding recombinant avian influenza virus HA protein, virus-like particle, vaccine, preparation and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Construction of the recombinant bacmid of embodiment 1 HA, NA and M1 gene

[0056] (1) Construction of HA, NA, and M1 gene recombination transfer plasmids

[0057] (1) In this embodiment, the nucleotide sequences of the HA, NA, and M1 genes of the avian influenza virus have been codon-optimized, biased towards insect cell expression, and 6x his tags are added to the C-terminals of the HA, NA, and M1 genes; Artificially synthesized to obtain the nucleotide sequences of the codon-optimized HA, NA, and M1 genes, and respectively link them into the PUC57 vector to obtain the corresponding recombinant plasmids (Beijing Liuhe Huada Gene Technology Co., Ltd.); wherein, the codon-optimized The nucleotide sequence of the HA gene is SEQ ID NO:1, and its amino acid sequence is SEQ ID NO:4; the nucleotide sequence of the NA gene after codon optimization is SEQ ID NO:2, and its amino acid sequence is SEQ ID NO:5; the nucleotide sequence of the M1 gene after codon optimization is SE...

Embodiment 2

[0078] The rescue of the recombinant baculovirus of embodiment 2 HA, NA and M1 gene

[0079] (1) Utilize conventional liposome-mediated transfection method, respectively the recombinant bacmid-HA, Bacmid-NA, Bacmid-M1 transfection sf9 insect cell (Invitrogen company) that embodiment 1 makes, at 27 Cultivate at ℃; when the cells were cultured for 72 hours, pathological changes appeared, and the cell culture supernatant was collected to obtain the first-generation recombinant baculovirus (P1) BV-HA, BV-NA, and BV-M1 respectively;

[0080] (2) Inoculate sf9 cells with the recombinant baculovirus of the P1 generation, and collect the cell supernatant (that is, the recombinant baculovirus of the P2 generation) when the cytopathic changes are obvious, and proceed to obtain the HA, NA, and M1 recombinant baculoviruses of the P3 generation in sequence. .

Embodiment 3

[0081] Example 3 Expression, optimization and purification of H7N9-VLP in insect cells

[0082] (1) Inoculate P3-generation HA, NA, and M1 recombinant baculoviruses into suspension-cultured High five cells (Invitrogen) at an MOI of 7:4:2, harvest the cells 96 hours after inoculation, and obtain the extracellular culture supernatant and cells after centrifugation. ;Cells were resuspended and crushed, and the broken supernatant in the cells was harvested by centrifugation; the hemagglutination titer of the virus-like particles in the extracellular culture supernatant was determined to be 11log2, and the hemagglutination titer of the broken supernatant in the cells was 13log2;

[0083] (2) Inoculate suspension-cultured High five cells (Invitrogen) with P3-generation HA, NA, and M1 recombinant baculoviruses at an MOI of 3:3:2, harvest the cells 96 hours after inoculation, and obtain extracellular culture supernatant and cells after centrifugation. The cells were resuspended and cr...

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Abstract

The invention belongs to the technical field of genetic engineering vaccines, and particularly relates to a gene for coding recombinant avian influenza virus HA protein, a virus-like particle, a vaccine, preparation and application. The gene has a nucleotide sequence as shown in SEQ ID NO: 1. The invention also provides an avian influenza virus-like particle, the avian influenza virus-like particle is assembled by HA protein, NA protein and M1 protein, and has a hemagglutination titer up to 13log2. The invention also provides an avian influenza virus-like particle vaccine containing the virus-like particle, the vaccine can provide complete clinical protection and significantly inhibit detoxification against lethal attack of homologous and wild H7N9 subtype highly pathogenic avian influenza viruses, and a new vaccine choice is provided for prevention and control of H7N9 subtype avian influenza.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering vaccines, in particular to a gene encoding recombinant avian influenza virus HA protein, a virus-like particle, a vaccine, and its preparation and application. Background technique [0002] Avian Influenza virus (AIV) is an enveloped, segmented, negative-strand RNA virus belonging to the Orthomyxoviridae family and the genus Influenza virus. Avian influenza is one of the severe infectious diseases of poultry. [0003] Avian influenza virus encodes a variety of proteins. HA (surface antigen hemagglutinin) protein is a membrane protein encoded by avian influenza virus. main target antigen. NA (neuraminic acid) protein is also a membrane protein, and its main function is to assist the release of progeny viruses from host cells. HA and NA proteins are the main antigenic components in the development of avian influenza vaccines. [0004] Vaccination is one of the most effective measures ...

Claims

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Application Information

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IPC IPC(8): C12N15/44C12N15/866C12N5/10A61K39/145A61P31/16
CPCC07K14/005C12N15/86A61K39/12A61P31/16C12N2760/16122C12N2760/16123C12N2760/16134C12N2710/14043C12N2800/22Y02A50/30
Inventor 樊惠英孔德鑫廖明陈陶然胡小龙
Owner SOUTH CHINA AGRI UNIV
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