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Construction and application of H9N2-subtype avian influenza virus cell high-yield vaccine strain

An avian influenza virus, avian influenza technology, applied in the direction of antiviral agents, viruses/phages, medical preparations containing active ingredients, etc., can solve the problems of chicken embryo virus residues, high production costs, environmental hazards, etc. Shortage, increased virus titer, good adaptability

Active Publication Date: 2016-06-15
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although chicken embryo inactivated vaccines have the advantages of convenient transportation, simple preparation process, safety and stability, there are many disadvantages in the cultivation of influenza virus in chicken embryos as follows: (1) when influenza outbreaks on a large scale, collect 9-10 days in a short period of time. It is very difficult for young chicken embryos
(2) Cultivate influenza virus with chicken embryos, it is easy to produce a large amount of discarded chicken embryos and virus residues, and discharge into nature is very harmful to the environment
(3) Affected by maternal antibodies; (4) Disadvantages such as high production cost and long cycle

Method used

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  • Construction and application of H9N2-subtype avian influenza virus cell high-yield vaccine strain
  • Construction and application of H9N2-subtype avian influenza virus cell high-yield vaccine strain
  • Construction and application of H9N2-subtype avian influenza virus cell high-yield vaccine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: A / chicken / Hubei / W1 / 2004 strain whole genome infectious clone of avian influenza virus isolate

[0045] 1. Extraction of RNA of avian influenza virus isolate A / chicken / Hubei / W1 / 2004

[0046] Take out the spare chicken embryo allantoic fluid avian influenza virus isolate A / chicken / Hubei / W1 / 2004 (referred to as virus) from the -80°C refrigerator, [see: Xiao-JuanXu, Gao-YuanXu, Hong-BoZhou, Zheng- JunYu.EvolutionarycharacterizationofinfluenzavirusA / duck / Hubei / W1 / 2004(H9N2)isolatedfromcentralChinaVirusGenes.2008Feb;36(1):79-83.Epub2007Nov20] Dissolve at room temperature, take 300μL of virus liquid and add it to the tube containing 1mL Trizol1.5mLEP, gently Gently turn it upside down, put it on ice for 15 minutes; then add 200 μL of chloroform, vortex and mix it with a shaker for 15 seconds, place it on ice for 10 minutes, centrifuge at 4 °C, 12000 r / min for 10 minutes, and take the supernatant (avoid suction) until white precipitate) into a new 1.5mLEP tube, a...

Embodiment 2

[0120] Example 2: Rescue of mutant strain W1-HA358 avian influenza virus and observation of its biological characteristics

[0121] 1. Extraction of endotoxin-free plasmid

[0122] Plasmids were extracted using the Endo-free Plasmid Mini Kit II produced by Omega. Specific steps are as follows:

[0123] (1) Inoculate the Escherichia coli liquids of eight constructed plasmids, pHW2000-PB1, pHW2000-PB2, pHW2000-PA, pHW2000-HA358, pHW2000-NA, pHW2000-NP, pHW200-M, pHW2000-NS, into 100mL of LB liquid medium, and then cultivate E. coli overnight at 37°C for 12-16h on a shaker.

[0124] (2) Add all 100mL of the bacterial solution into a 50mL centrifuge tube, balance, centrifuge at 12000r / min for 8min at 4°C, discard the culture supernatant, and collect the bacteria.

[0125] (3) Discard the supernatant, add an appropriate amount of normal saline or PBS to resuspend the bacteria in the pellet, aliquot into 2 mL EP tubes, centrifuge at 12000 r / min at 4°C for 2 min, discard the cultu...

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Abstract

The invention belongs to the technical field of animal vaccine preparation and particularly relates to construction and application of an H9N2-subtype avian influenza virus cell high-yield vaccine strain. The cell vaccine strain is prepared through artificially induced mutation screening of an avian influenza isolate and is named H9N2-subtype avian influenza virus W1-HA358 strain, of which an original isolate is A / duck / Hubei / W1 / 2004 (H9N2) strain. The 3rd, 5th and 8th loca of a 3'-terminal of an HA gene non-coding region of the isolate are subjected to artificial site-specific mutagenesis, and a mutation strain is saved through reverse genetic operation, wherein the mutation strain is subjected to passage on an MDCK cell until a stable generation to obtain a high-proliferation-titer virus strain, thereby producing the H9N2-subtype avian influenza virus cell vaccine. The invention solves the problem of instable proliferation of the H9N2-subtype avian influenza on the MDCK cell, and overcomes a defect of shortage of large quantity of chick embryo during outbreak of the avian influenza, thereby increasing preparation efficiency of the vaccine.

Description

technical field [0001] The invention belongs to the technical field of animal vaccine preparation. It specifically relates to the construction and application of a high-yield vaccine strain of H9N2 subtype avian influenza virus cells. Background technique [0002] H9N2 subtype avian influenza (Avianinfluenza, AI) is an infectious syndrome caused by type A influenza virus infecting poultry, which spreads widely around the world and seriously threatens the development of animal husbandry. In 1966, the H9N2 subtype AIV was detected from turkey for the first time. Since then, AIV has been widely spread in poultry. In 1994, my country reported for the first time that H9N2 subtype AIV was isolated from a chicken farm in Guangdong, and since then H9N2 subtype AIV has been widely spread in poultry farming areas in my country. Although H9N2 subtype AIV belongs to low pathogenicity AIV, studies now show that it can also infect mice, ferrets and humans. In addition, it can cause a d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/145A61P31/16
Inventor 周红波金梅林白绕仙王玉刚李国利康超阳姹
Owner HUAZHONG AGRI UNIV
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