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A kind of artificially recombined h9n2 avian influenza virus strain, preparation method, vaccine composition and application thereof

An avian influenza virus and virus strain technology, applied in the field of reverse genetics technology and animal infectious diseases, can solve the problems of poor cross protection, difficulty in screening H9N2 virus strain vaccine, time-consuming and laborious, etc., and achieve good immunogenicity and good chicken embryos. adaptive effect

Active Publication Date: 2018-05-04
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The strains within the H9N2 subtype are quite different, and it is difficult to screen out H9N2 strains with broad-spectrum protection as vaccines;
[0008] The traditional method to obtain recombinant strains is to co-infect chicken embryos or cells with several different strains, and screen the recombinant strains. This recombination method is time-consuming and laborious; and the HA and NA of some recombinant viruses using reverse genetics come from the same strain, poor cross protection

Method used

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  • A kind of artificially recombined h9n2 avian influenza virus strain, preparation method, vaccine composition and application thereof
  • A kind of artificially recombined h9n2 avian influenza virus strain, preparation method, vaccine composition and application thereof
  • A kind of artificially recombined h9n2 avian influenza virus strain, preparation method, vaccine composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Cloning of related genes

[0039] 1. Extraction of RNA of PR8 influenza virus (obtained from the depository institution of St. Jude Children's Research Hospital)

[0040] Avian influenza virus RNA was extracted with reference to the instructions of the Viral Nucleic Acid Extraction Kit II kit (made by Taiwan Xuji Company (Geneaid Biotech)), and the specific method was as follows:

[0041] 1) Take 200 μL (if insufficient, make up 200 μl with PBS) sample into a 1.5ml EP tube, add 400 μL VB lysate, mix well with a vortexer, and incubate at room temperature for 10 minutes.

[0042] 2) Add 450 μL of AD solution (make sure absolute ethanol has been added to the AD solution), and shake vigorously.

[0043] 3) Put the VB membrane into a 2ml collection tube, add 600 μL of the above lysis mixture to the VB membrane, and centrifuge at 14000-16000 g for 1 min.

[0044] 4) Discard the liquid in the collection tube, put the VB membrane back into the collection tube, add ...

Embodiment 2

[0102] Example 2: Rescue and biological characteristics of recombinant H9N2 avian influenza virus

[0103] 1. Extraction of endotoxin-free plasmid

[0104] Extract according to the steps of the kit Endo-free plasma mini kit II provided by OMEGA (operate according to the instructions of the kit), the specific steps are as follows:

[0105] (1) Escherichia coli carrying the target plasmid was inoculated into a 10-20ml culture tube containing 5ml ampicillin LB, and cultured on a shaker at 37°C for 12-16h to amplify the plasmid. Use a 10-20ml culture tube or flask with at least 4 times the volume of the culture medium.

[0106] (2) Take 5.0ml of the bacterial solution and centrifuge at 10000g for 1min at room temperature to precipitate the bacterial species.

[0107] (3) Pour out or suck out the above-mentioned LB medium and discard it. Add 250 μL solution I / RNase A mixture to the pellet to resuspend the bacteria, and vortex to completely resuspend the cells.

[0108] (4) Add ...

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Abstract

The invention provides an H9N2 avian influenza virus strain, which is rH9N2‑2 / 6 virus strain recombined with HA protein of SEQ. NO. 2 protein sequence and NA protein of SEQ ID NO: 4 protein sequence. The recombinant H9N2 avian influenza virus rH9N2-2 / 6 strain was rescued by reverse genetics technology, which has good immunogenicity. 28 days after immunization with the inactivated vaccine prepared by this virus strain, the HI antibody reached 11Log2. The reverse genetics technology of the present invention can quickly obtain the target virus, and screen out and prepare a vaccine that can prevent broad-spectrum viruses, and the vaccine can provide complete protection against H9N2 attack after immunization.

Description

technical field [0001] The invention relates to the technical fields of reverse genetics technology and animal infectious disease, in particular to an artificial recombinant H9N2 bird flu vaccine and its application. Background technique [0002] Avian influenza virus (Avian influenza virus AIV) is an important pathogen of the respiratory system of birds and some mammals, and belongs to the genus Influenzavirus of the family Orthomyxoviridae in taxonomy. [0003] According to the different surface protein antigenicity, avian influenza virus is divided into 17 subtypes of hemagglutinin (HA) and 10 subtypes of neuraminidase (NA), among them, the H9N2 subtype of avian influenza virus is widespread in the world The epidemic has not only caused huge economic losses to the poultry industry, but also caused danger to human health. my country isolated the first strain of H9N2 subtype avian influenza from a chicken farm in Guangdong Province in 1994. Since then, this subtype of avia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/85A61K39/145A61P31/16C07K14/11C12R1/93
Inventor 张许科孙进忠王同燕田克恭
Owner PU LIKE BIO ENG
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