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H9N2 subtype avian influenza virus strain as well as inactivated vaccine and application thereof

A technology of avian influenza virus and inactivated vaccine, which is applied in the field of isolation and application of avian influenza virus strains, and can solve problems such as high cost, large batch-to-batch variation in product quality, and infection of chicken-derived potential diseases.

Active Publication Date: 2014-07-23
JILIN ZHENGYE BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the vast majority of commercially available avian influenza vaccines are chicken embryo tissue seedlings, which have disadvantages such as high cost, large batch-to-batch difference in product quality, and easy infection of chicken-derived potential diseases compared with cell-based vaccines.

Method used

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  • H9N2 subtype avian influenza virus strain as well as inactivated vaccine and application thereof
  • H9N2 subtype avian influenza virus strain as well as inactivated vaccine and application thereof
  • H9N2 subtype avian influenza virus strain as well as inactivated vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1H9

[0025] Isolation and identification of embodiment 1H9N2 subtype avian influenza virus J / ZY strain

[0026] 1. Experimental method

[0027] 1.1 Isolation of virus

[0028] In 2010, from chickens suspected of H9N2 avian influenza in a chicken breeding area in Jilin Province, China, the lungs and livers of sick chickens with typical clinical symptoms were aseptically cut into pieces, and added sterilized at a ratio of 1:5 (W / V). After bacterial normal saline and mortar grinding, freeze-thaw three times at -40°C / room temperature, centrifuge at 3000r / min for 15min, and take the supernatant for later use.

[0029] The supernatant of the above disease material was sterilized through a 0.22 μm microporous filter, and the allantoic cavity was inoculated with 10-day-old non-immune chicken embryos. Eight chicken embryos were inoculated with each disease material, and each chicken embryo was inoculated with 0.2 mL. At the same time, it was used as a control without inoculation, and incu...

experiment example 1

[0043] Pathogenicity determination of the H9N2 subtype avian influenza virus J / ZY strain isolated in Experimental Example 1

[0044] 1. Experimental method

[0045] 1.1 Proliferation of virus J / ZY strain in MDCK cells

[0046] After diluting the purified tissue poison with sterilized normal saline, inoculate 90% of MDCK cells grown into a monolayer, culture at 37°C for 48-72h, and the cytopathic rate reaches more than 75%, put it in -70°C and freeze-thaw three times repeatedly , to harvest virus fluid. Passage 10 times until the virus proliferates well in MDCK cells.

[0047] 1.2 Virus EID 50 determination

[0048] Make a 10-fold gradient dilution of the isolated virus with sterilized normal saline, set 10 -1 ~10 -9 9 dilution gradients, and set up a blank control group, inoculate five 10-day-old non-immune chicken embryos for each dilution, discard the 24-hour dead embryos, freeze the chicken embryos overnight at 4°C 72 hours after inoculation, harvest the allantoic flu...

experiment example 2H9

[0059] Experimental example 2 The immune protection experiment of H9N2 subtype avian influenza virus J / ZY strain

[0060] 1. Experimental method

[0061] 1.1 Amplification of virus

[0062] The isolated virus J / ZY strain was amplified by MDCK cell roller bottle culture (collecting the cell culture supernatant) and inoculated with SPF chicken embryos (collecting the allantoic fluid). Separately, the same day-old and the same batch of SPF inoculated with sterile PBS were set up. The allantoic fluid of chicken embryos was harvested as normal blank chicken embryo allantoic fluid. The above-mentioned amplified virus fluids were collected and evenly distributed, and stored at -70°C for later use.

[0063] 1.2 Preparation of inactivated vaccine

[0064] The two virus liquids amplified by MDCK cells and SPF chicken embryos were centrifuged at 5000r / min for 30min, the supernatant was taken, added formalin solution with a final concentration of 0.1%, and inactivated at 37°C for 48h. ...

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Abstract

The invention discloses an H9N2 subtype avian influenza virus strain as well as an inactivated vaccine and application thereof, and belongs to the field of separation and application of avian influenza virus strains. The H9N2 subtype avian influenza virus strain J / ZY strain is separated from a suspected H9N2 avian influenza pathogenetic chicken flock, and the microbial collection number of the strain is CGMCC No. 8853. The invention further discloses a preparation method for the H9N2 subtype avian influenza inactivated vaccine, and the preparation method comprises the following steps: culturing viruses to obtain a virus supernate; adding an inactivating agent to inactivate the virus supernate; mixing adjuvants and the virus supernate uniformly, and emulsifying to obtain the inactivated vaccine. Immune protection experiments prove that the separated H9N2 subtype avian influenza virus strain J / ZY strain serving as a vaccine strain has good immunogenicity and a relatively high antibody level can be produced after an animal is immunized by the strain.

Description

technical field [0001] The present invention relates to avian influenza virus strains, in particular to an isolated H9N2 subtype avian influenza virus (Avain Influenza Virus) J / ZY strain and an inactivated vaccine for preventing H9N2 subtype avian influenza virus prepared by using the strain, belonging to Isolation and application fields of avian influenza virus strains. Background technique [0002] Avian influenza (abbreviated as avian influenza, Avain Influenza, AI) is an infection and / or disease syndrome of avian (including poultry and wild fowl) caused by type A influenza virus, mainly causing systemic or respiratory systemic The disease was established as a Class A infectious disease by the World Organization for Animal Health. According to the different pathogenicity of avian influenza, avian influenza can be divided into highly pathogenic avian influenza, low pathogenic avian influenza and apathogenic avian influenza. [0003] H9N2 subtype avian influenza belongs t...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/145A61P31/16C12R1/93
Inventor 何玉友冯建民李彥伟吴艳丽张闯田丽华王石王国辉刘斌杨键李长艳廉维杨泽彬柳志光冯会白杨
Owner JILIN ZHENGYE BIOLOGICAL PROD
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