General type DNA vaccine for influenza A, and preparation method and application of general type DNA vaccine for influenza A

A DNA vaccine, influenza A technology, applied in the fields of biopharmaceuticals and genetic engineering, to achieve the effect of easy storage and transportation, high safety and simple operation

Pending Publication Date: 2020-08-11
HENAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • General type DNA vaccine for influenza A, and preparation method and application of general type DNA vaccine for influenza A
  • General type DNA vaccine for influenza A, and preparation method and application of general type DNA vaccine for influenza A
  • General type DNA vaccine for influenza A, and preparation method and application of general type DNA vaccine for influenza A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Preparation and Identification of 3 Kinds of Recombinant Plasmids

[0045] 1. Materials

[0046] Main reagents: E.coli XL10-Gold competent cells were prepared and preserved by our laboratory, gene synthesis was provided by Jinweizhi Company, technical services such as recombinant plasmid gene sequencing and primer synthesis were provided by Jinweizhi Company. Endonucleases were purchased from Thermo Company, antibiotics, etc. were purchased from Suleibao Company, HA rabbit polyclonal antibody was purchased from Beijing Yiqiao Sino Biological Company, M2 mouse monoclonal antibody, and NP mouse monoclonal antibody were purchased from abcam Company.

[0047] 2. Method

[0048] 2.1 Extract RNA from H5N1 virus and then reverse transcribe it into cDNA, use the primers of HAF and HAR of H5N1 respectively with cDNA as template, SEQ ID NO.9, SEQ ID NO.10 are the amplification primers of HA fragment, carry out PCR amplification Increase (94°C for 5 minutes, 94°C for 3...

Embodiment 2

[0059] Example 2 Influenza A Universal DNA Vaccine Experimental Immunization Research

[0060] 1. Materials

[0061] HPR-labeled goat anti-mouse IgG, IgG1, and IgG2a were purchased from Southern Biotechnology, ELISApot detection kits for mouse IFN-γ and IL-4 were purchased from Mabtech, and TMB was purchased from Sigma. BALB / c female mice aged 6-8 weeks were purchased from Beijing Weitong Lihua Company.

[0062] 2. Method

[0063] 2.1 Immunity and attack

[0064] For immune challenge design, 180 healthy female Balb / c mice were randomly divided into 6 immunization groups, 30 mice in each group, and the experimental groups were immunized with pcDNA 3.1-HA+adjuvant (CpG+MPLA), pcDNA3.1 -mHA+adjuvant (CpG+MPLA), pcDNA 3.1-cHA+adjuvant (CpG+MPLA), pcDNA3.1-sHA+adjuvant (CpG+MPLA), control group immune pcDNA 3.1+adjuvant group (CpG+MPLA), The blank group is the PBS group. A total of 3 immunizations were performed, all of which were injected into the tibialis anterior muscle of ...

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Abstract

The invention provides a general type DNA vaccine for influenza A, and a preparation method and application of the general type DNA vaccine for influenza A. Genes of three fusion proteins are constructed through highly-conservative sequences of an HA protein, an M2e protein and a NP protein of influenza viruses, then the genes are cloned to a pCDNA3.1(+) carrier, and three recombinant DNA vaccinespcDNA3.1-sHA, pcDNA3.1-mHA and pcDNA3.1-cHA are constructed. The DNA vaccine can be used for respects of preventing influenza homologous viruses, influenza heterogenesis viruses and the like of lactation type wild animals of tigers and the like. The DNA vaccine disclosed by the invention is simple in preparation method, low in cost, good in immunogenicity and good in cross protection, can meet preparation requirements for large-scale influenza vaccinations, is food in biological safety, and can arouse immunity of organisms to various influenza viruses.

Description

technical field [0001] The invention relates to the fields of genetic engineering, microorganisms and biopharmaceuticals, in particular to a universal influenza A DNA vaccine and its preparation method and application. Background technique [0002] Influenza virus can cause human and animal infection, and influenza pandemic is a disaster that brings great losses to human health and social economy. It has the characteristics of short incubation period, rapid onset, rapid transmission, and many complications. In the past 100 years, there have been four influenza pandemics that have infected hundreds of millions of people and killed tens of millions. Since the Spanish flu pandemic in 1918, British scientists isolated the first H1N1 influenza virus strain in 1933, which greatly accelerated human understanding of influenza viruses. In the 1940s, influenza vaccines appeared for the first time, which enabled humans to prevent and control influenza viruses. A pandemic became possi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145A61P31/16C12N15/62C12N15/44
CPCA61K39/12A61K2039/55561A61K2039/55572A61K2039/572A61K2039/575A61P31/16C07K14/005C07K2319/02C12N2760/16122C12N2760/16134
Inventor 任志广高玉伟夏咸柱窦悦杨争艳王铁成陈明涛赵永坤李元果冯娜杨松涛
Owner HENAN UNIVERSITY
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