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A kind of preparation method of highly pathogenic h7n9 avian influenza virus, vaccine, detection reagent and virus, vaccine

An avian influenza virus, highly pathogenic technology, applied in antiviral agents, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of insufficient protection, low safety, and low vaccine immunogenicity. question

Inactive Publication Date: 2021-07-06
GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to overcome the deficiencies in the prior art, one of the purposes of the present invention is to provide a preparation method of highly pathogenic H7N9 avian influenza virus, by using the virus to prepare vaccines, to overcome the preparation of traditional highly pathogenic H7N9 avian influenza virus vaccines Low process safety, low immunogenicity of the prepared vaccine, unable to provide sufficient protection, etc.
[0008] The second object of the present invention is to provide a highly pathogenic H7N9 avian influenza virus, by using the virus to prepare vaccines, to solve the problems of traditional vaccine preparation process biosafety and low vaccine immunogenicity
[0009] The third object of the present invention is to provide a highly pathogenic H7N9 avian influenza vaccine to solve the problems that traditional vaccines cannot provide sufficient protection against the infection of highly pathogenic H7N9 avian influenza virus

Method used

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  • A kind of preparation method of highly pathogenic h7n9 avian influenza virus, vaccine, detection reagent and virus, vaccine
  • A kind of preparation method of highly pathogenic h7n9 avian influenza virus, vaccine, detection reagent and virus, vaccine
  • A kind of preparation method of highly pathogenic h7n9 avian influenza virus, vaccine, detection reagent and virus, vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Preparation method of highly pathogenic H7N9 avian influenza virus with high immunogenicity

[0053] The first step is to synthesize the HA gene:

[0054] First, according to the HA gene sequence of the highly pathogenic H7N9 avian influenza virus vaccine strain A / Guangdong / 17SF003 / 2016 (H7N9) recommended by WHO, synthesize the HA gene with multiple basic amino acid deletions at the cleavage site, and multiple bases at the cleavage site The HA gene (referred to as H7 / GD16 / WT) with sex amino acid deletion was completed by GenScript. Existing studies have shown that the cleavage site with multiple basic amino acids is a sign of highly pathogenic avian influenza virus, and the deletion of multiple basic amino acids can reduce its pathogenicity, so that the recombinant virus can be tested in the biosafety level two experiment. The operation is carried out in the room without affecting the immunogenicity of the HA protein. For safety reasons, the cleavage site in the HA ge...

Embodiment 2

[0069] The difference between Example 2 and Example 1 is that the NA fragment in Example 2 is selected from the NA gene of the N9 subtype without oseltamivir resistance mutation. The N9 subtype NA gene containing the oseltamivir resistance mutation has the effect of oseltamivir resistance, and the highly immunogenic and highly pathogenic H7N9 avian influenza virus prepared by using the mutated NA gene and the corresponding The vaccine has the risk of causing the proliferation of drug resistance genes. By using non-oseltamivir drug resistance mutated NA genes, the widespread dissemination of oseltamivir drug resistance mutated NA genes can be avoided. The synthesis of HA gene of H7 / GD16 / WT, the construction of pM-H7 / GD16 / WT recombinant plasmid and the site-directed mutation of HA gene in Example 2 are all the same as in Example 1 above.

[0070] The first step, NA gene synthesis:

[0071] The NA gene was selected from the NA gene (GISAID ID: EPI439509, called N9 / AH13) of the l...

Embodiment 3

[0084] The preparation method of highly pathogenic H7N9 avian influenza inactivated vaccine with high immunogenicity comprises an inactivation step and a purification step.

[0085] The first step, inactivation of influenza virus

[0086] The two recombinant influenza viruses prepared in Example 2 above were inactivated, and the specific inactivation method was as follows. Formaldehyde solution was used for inactivation, and formaldehyde solution with a final concentration of 0.1% was added to the virus allantoic fluid, and inactivated at 37° C. for 16 hours. The inactivated influenza virus was continuously passaged in chicken embryos for three times, and it was determined that the inactivation was successful if no hemagglutination titer was detected after passage.

[0087] The second step, the purification of influenza virus

[0088] The inactivated influenza virus chicken embryo allantoic fluid was centrifuged at 120000×g for 1.5 h in an ultracentrifuge to concentrate the ...

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Abstract

The invention discloses a preparation method of a highly pathogenic H7N9 avian influenza virus, which can improve the immunogenicity of the virus, comprising the following steps: preparing a Q226L mutant HA gene, and preparing a highly pathogenic H7N9 avian influenza virus with a Q226L mutation The gene sequence of the full-length HA protein is obtained to obtain the Q226L mutant HA gene; the virus is rescued, and the recombinant influenza virus is rescued by using the Q226L mutant HA gene. The invention also discloses the highly pathogenic H7N9 avian influenza virus prepared by the method, the vaccine prepared by using the highly pathogenic H7N9 avian influenza virus and its preparation method, and the detection method prepared by using the highly pathogenic H7N9 avian influenza virus reagent. The preparation method of the highly pathogenic H7N9 avian influenza virus of the invention improves the biological safety and immunogenicity of the recombinant virus.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to the field of virus genetic engineering technology, in particular to a highly pathogenic H7N9 avian influenza virus and a preparation method thereof, and the invention also relates to a vaccine prepared by using the highly pathogenic H7N9 avian influenza virus As well as the preparation method of the vaccine, the present invention also relates to a detection reagent prepared by using the highly pathogenic H7N9 avian influenza virus. Background technique [0002] Influenza A virus (Influenza A virus) belongs to the Orthomyxoviridae family, and the virus is mainly spherical with an outer envelope. The influenza A virus genome is 8-segment single-stranded negative-sense RNA, encoding more than 12 kinds of proteins, among which the main glycoproteins on the surface of the envelope are hemagglutinin protein (HA protein) and neuraminidase (NA). According to the difference of HA pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N15/44C12N15/85A61K39/145A61P31/16G01N33/569
CPCA61K39/12A61K2039/5252A61P31/16C12N7/00C12N15/85C12N2760/16121C12N2760/16122C12N2760/16134G01N33/56983G01N2469/20
Inventor 王洋陈凌潘蔚绮吕云华董记
Owner GUANGZHOU MEDICAL UNIV
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