Recombinant baculovirus expressing manually modified and synthesized influenza A H1N1 virus HA-NA-M1 gene

A technology of influenza virus and type A, applied in the field of virology, can solve the problems of ineffective prevention of new influenza viruses and increased disease risk, and achieve the effects of easy monitoring, increased expression level, and improved screening efficiency

Inactive Publication Date: 2010-01-13
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the most effective way to treat influenza A (H1N1) virus is antiviral drug therapy. At present, oseltamivir (also known as Tamiflu) and zanamivir are commonly used, but anti-Tamiflu drugs have been found in Denmark, Japan, the United States, Hong Kong, China and other countries and regions. resistant strains ( http://www.who.int/csr/disease/swineflu/notes/h1n1_antiviral_resistance_20090708/en/index.html ), thus increasing the risk of the disease, the field is

Method used

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  • Recombinant baculovirus expressing manually modified and synthesized influenza A H1N1 virus HA-NA-M1 gene
  • Recombinant baculovirus expressing manually modified and synthesized influenza A H1N1 virus HA-NA-M1 gene
  • Recombinant baculovirus expressing manually modified and synthesized influenza A H1N1 virus HA-NA-M1 gene

Examples

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Embodiment 1

[0022] Example 1: Construction of a recombinant baculovirus QP-Ac-HNM1 expressing the artificially modified and synthesized influenza A H1N1 virus HA-NA-M1 gene.

[0023] 1. Synthesis of main immunogenic genes of H1N1 influenza virus.

[0024] Referring to the HA, NA and M1 gene sequences of the H1N1 influenza virus A / California / 04 / 2009 (H1N1) strain, the sequences of the HA, NA and M1 genes were artificially synthesized according to the codon preference, wherein the NA and M genes were synthesized Together, the promoter (PH) of the baculovirus polyhedron gene is added at the N end of the NA gene, the poly(A) sequence of the SV40 virus is introduced at its C end, and the promoter PH is added at the N end of the M1 gene.

[0025] 2. Construction of the baculovirus transfer vector pFBDPHmHNM1P10eGFP.

[0026] According to the backbone of the baculovirus vector pFastBacDual in the Bac-to-Bac Baculovirus Expression System (kit) of Invitrogen, the polyA of chicken globin is insert...

Embodiment 2

[0034] Example 2: Identification of recombinant baculovirus QP-Ac-HNM1, preparation of VLP vaccine and immunogenicity experiment.

[0035] 1. Identification of recombinant baculovirus QP-Ac-HNM1.

[0036] The sf9 cells were inoculated into 6-well cell culture plates, and when the cells grew to 80-90% monolayer, they were inoculated with 1 MOI of recombinant baculovirus QP-Ac-HNM1, and a control baculovirus was set at the same time, and observed daily. After 72 hours of infection, more than 90% of sf9 cells glowed green fluorescence.

[0037] Take 400 μl of culture supernatant and cell suspension, treat with trypsin at 56°C for 4 hours, extract twice with an equal volume of phenol / chloroform, take the supernatant and add 1 / 10 volume of 3M sodium acetate and 2 times volume of Precipitate with absolute ethanol, centrifuge to discard the supernatant, wash the precipitate once with 500 μl 70% ethanol, dry it naturally, add 50 μl sterilized deionized water to resuspend and use it a...

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Abstract

The invention relates to the field of virology, in particular to a recombinant baculovirus which is manually modified and synthesized and contains a main immunogenic gene HA-NA-M1 of an influenza A H1N1 virus. The strain QP-Ac-HNM1 belongs to the baculovirus (Baculovirus) and is preserved in the China Center for Type Culture Collection (CCTCC) with the preserving number of CCTCC-V200912. The recombinant virus is capable of synchronously expressing the HA and NA of the influenza A H1N1 virus and M1 proteins to form virus particles which can be used for developing vaccines so as to prevent human beings and swine from being infected with the influenza A H1N1 virus.

Description

technical field [0001] The present invention relates to the field of virology. More specifically, the present invention relates to a new recombinant baculovirus strain containing artificially modified and synthesized main immunogenic gene HA-NA-M1 and marker gene eGFP of Influenza A H1N1 virus. This recombinant strain marker gene eGFP and H1N1 influenza virus HA-NA-M1 protein, and packaged into influenza-like virus particles. The vaccine developed by the virus particles can prevent the infection of human and swine type A H1N1 influenza virus. Background technique [0002] Influenza A H1N1 (Influenza virus H1N1) is one of the important primary pathogens that cause respiratory diseases in humans and pigs, and is often mixed with other pathogens to cause more serious losses. The virus belongs to the Orthomyxoviridae Influenza virus genus, and is divided into three types A, B and C according to the antigenicity of the virus nucleoprotein (internationally referred to as A, B an...

Claims

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Application Information

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IPC IPC(8): C12N7/01C07K14/11A61K48/00A61K39/145A61P31/16C12R1/93
Inventor 钱平李祥敏金梅林陈焕春
Owner HUAZHONG AGRI UNIV
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