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Method for producing inactivated vaccine of H9N2 subtype of avian influenza virus and product of inactivated vaccine

A production method and technology of inactivated vaccines, applied in the direction of medical preparations containing active ingredients, pharmaceutical formulas, antibody medical components, etc., can solve problems such as the production of H9N2 subtype avian influenza inactivated vaccines, and meet the requirements of immunization production, Effect of reducing contamination risk and saving cultivation space

Inactive Publication Date: 2015-02-11
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the use of cell factories to produce inactivated vaccines for H9N2 subtype avian influenza

Method used

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  • Method for producing inactivated vaccine of H9N2 subtype of avian influenza virus and product of inactivated vaccine
  • Method for producing inactivated vaccine of H9N2 subtype of avian influenza virus and product of inactivated vaccine
  • Method for producing inactivated vaccine of H9N2 subtype of avian influenza virus and product of inactivated vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036]Example 1 Isolation and Identification of H9N2 Subtype Avian Influenza Virus NJ13-8 Strain

[0037] 1. Experimental method

[0038] 1.1 Isolation of virus

[0039] In 2013, the lungs and livers were collected from a chicken farm in Nanjing, Jiangsu Province, and sent for inspection. The lungs and livers were cut into pieces, added with sterilized saline at a ratio of 1:5, ground in a sterile mortar, frozen and thawed three times repeatedly, and subjected to 3000r / min centrifuged for 15min, and the supernatant was taken for later use.

[0040] The supernatant of the above-mentioned disease material was sterilized through a 0.22 μm disposable filter, and inoculated with 10-day-old SPF chicken embryos through the allantoic cavity, with 0.1 ml for each chicken embryo, and a total of 5 chicken embryos were inoculated. Incubate at 37°C; discard dead embryos within 24 hours, aseptically collect the allantoic fluid of chicken embryos that died within 24-120 hours, and store a...

Embodiment 2

[0067] Example 2 Pathogenicity assay of isolated virus NJ13-8 strain and its proliferation in MDCK cells

[0068] 1. Experimental method

[0069] 1.1 Pathogenicity determination of isolated virus NJ13-8 strain

[0070] Embryo median infectious dose (EID) 50 ), intracerebral pathogenicity index (ICPI) and intravenous inoculation index (IVPI) test to detect the pathogenicity of the virus NJ13-8 strain that embodiment 1 isolates.

[0071] 1.1.1 Virus EIDs 50 determination

[0072] Make the isolated virus with sterilized saline for 10 -6 ~10 -9 Dilution, and set up a blank control group, inoculate 5 10-day-old SPF chicken embryos at each dilution, discard 24h dead embryos, freeze the chicken embryos overnight at 4°C 120h after inoculation, harvest chicken embryo allantoic fluid, and measure its HA efficacy Valence, and refer to the Reed-Muench method to calculate the virus EID 50 .

[0073] 1.1.2 Determination of intracerebral pathogenicity index (ICPI)

[0074] Take 10 1...

Embodiment 3

[0099] Example 3 Preparation of Inactivated Vaccine Using Cell Factory to Cultivate H9N2 Subtype Avian Influenza Virus

[0100] 1. Experimental method

[0101] 1.1 Cell culture

[0102] First, resuscitate MDCK cells into T75 cell flasks, grow for about 48 hours, and pass passage according to the ratio of 1:4; then transfer the cells in 6 T75 cell flasks full of cells to a 10L spinner bottle for culture; about 48 hours Subculture the cells at a ratio of 1:3; the medium used in this process is DMEM, the serum is fetal bovine serum, and the usage amount is 10%.

[0103] The cells obtained in the spinner bottle were washed twice with PBS, and then 125 ml of 0.25% trypsin-EDTA solution was added for digestion, and the cells in the spinner bottle were harvested.

[0104] After the harvested cells were counted, the MDCK cells were cultured in the Thermo Scientific Nunc cell factory, and the medium used was DMEM medium. Samples were taken at 12h, 24h, and 48h after cell inoculation...

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Abstract

The invention discloses a method for producing an inactivated vaccine of H9N2 subtype of avian influenza virus and a product of the inactivated vaccine, and belongs to the field of production and application of inactivated vaccines of H9N2 subtype of avian influenza virus. The method comprises the following steps: (1) culturing a MDCK cell by utilizing a cell factory; (2) inoculating H9N2 subtype of avian influenza virus, and proliferating virus in the cell factory, wherein the microbial preservation number of the H9N2 subtype of avian influenza virus is CGMCC No.9325; and (3) harvesting the virus, and preparing the inactivated vaccine. Immune protection tests prove that the protective rate of the H9N2 subtype of avian influenza virus produced by the method is 100 percent, and the antibody level of a cytotoxicity immunity group at each point in time is higher than that of a germ toxicity immunity group, and rises relatively high. Virus liquid produced by the method can be used for preparing medicines or reagents for preventing or treating H9N2 subtype of avian influenza virus.

Description

technical field [0001] The invention relates to a production method of an H9N2 subtype avian influenza inactivated vaccine, in particular to a method for producing an H9N2 subtype avian influenza inactivated vaccine using a cell factory, and also relates to the H9N2 subtype avian influenza inactivated vaccine prepared by the production method. The live vaccine and its use in preparing medicines or reagents for preventing or treating H9N2 subtype avian influenza belong to the technical field of production and application of H9N2 subtype avian influenza inactivated vaccines. Background technique [0002] Avian Influenza (Avain Influenza, AI), abbreviated as Avian Influenza, is an infection and / or disease syndrome of birds (including poultry and wild birds) caused by Type A influenza virus, mainly causing systemic or respiratory system infection in birds. The disease was established as a Class A infectious disease by the World Organization for Animal Health. H9N2 subtype avian...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145A61P31/16
Inventor 李银刘宇卓李彥伟滕颖黄欣梅赵冬敏韩凯凯杨婧
Owner JIANGSU ACAD OF AGRI SCI
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