A kind of preparation method of avian influenza virus h9 subtype inactivated vaccine

An avian influenza virus and inactivated vaccine technology, which is applied in the field of preparation of avian influenza virus H9 subtype inactivated vaccines, can solve the problems of constant downstream technology, low virus titer, high production cost, etc. Vaccine effect, high stability effect

Active Publication Date: 2020-06-23
广州渔跃生物技术有限公司 +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above-mentioned avian influenza H9N2 subtype virus strains use SPF chicken embryo vectors for virus propagation, and the virus titer is not high, and the production cost of large-scale production with SPF chicken embryo vectors is very expensive, which is not conducive to large-scale production
[0005] At the same time, inoculation of influenza virus through the allantoic cavity of chicken embryos will occur. For example, the supply of vaccines during a pandemic is often limited by the number of SPF chicken embryos; some strains cannot replicate in chicken embryos, so it is difficult to obtain a sufficient amount of virus, only chicken Embryo-adapted strains can obtain high-yield viruses; mutations in receptor-binding specific sites lead to reduced antigenicity; the downstream technology of vaccine production is immutable and very heavy, easy to pollute, etc., which greatly limits the promotion and application of avian influenza H9 subtype vaccines

Method used

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  • A kind of preparation method of avian influenza virus h9 subtype inactivated vaccine
  • A kind of preparation method of avian influenza virus h9 subtype inactivated vaccine
  • A kind of preparation method of avian influenza virus h9 subtype inactivated vaccine

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Effect test

Embodiment 1

[0041] Embodiment 1, a kind of preparation method of avian influenza virus H9 subtype inactivated vaccine

[0042] Preparation of S1 cell carrier: Digest and disperse LMH cells with trypsin, and use DMEM medium containing 5% newborn bovine serum and 1000 units / mL penicillin-streptomycin double antibody at 37°C, 5% CO 2 LMH cells were cultured for 2 days under certain conditions, and then the cells were washed twice with serum-free DMEM medium to obtain cell carriers; the LMH cells were cultured in spinner bottles or placed in microcarrier reactors.

[0043] Inoculation of S2 virus: Inoculate the H9 subtype of avian influenza virus into the cell carrier obtained in step S1 according to the final volume of 1:60, place it at 37°C for 30 minutes, and then suck out the virus liquid, then add medium A, and incubate at 37°C , 5%CO 2 48h under the condition of culturing, 70% of the cells were damaged, and the diseased cells were obtained;

[0044] Described medium A comprises DMEM b...

Embodiment 2

[0052] Embodiment 2, a kind of preparation method of avian influenza virus H9 subtype inactivated vaccine

[0053] Preparation of S1 cell carrier: Digest and disperse LMH cells with trypsin, and use DMEM medium containing 8% newborn bovine serum and 1600 units / mL penicillin-streptomycin double antibody at 37°C, 5% CO 2 The conditions were cultivated for 2 days, and then the cells were washed 3 times with serum-free DMEM culture medium to obtain cell carriers; the LMH cells were cultured in a microcarrier reactor, and the amount of microcarriers used was 6 g / L.

[0054] Inoculation of S2 virus: Inoculate the H9 subtype of avian influenza virus into the cell carrier obtained in step S1 according to the final volume of 1:120, place it at 37°C for 40 minutes, then suck out the virus liquid, then add medium A, and incubate at 37°C , 5%CO 2 Under the conditions of 56 hours, 75% of the cells were damaged, and the diseased cells were obtained;

[0055] Described medium A comprises D...

Embodiment 3

[0063] Embodiment 3, a kind of preparation method of avian influenza virus H9 subtype inactivated vaccine

[0064] Preparation of S1 cell carrier: Digest and disperse LMH cells with trypsin, and incubate at 37°C, 5% CO with DMEM medium containing 10% newborn bovine serum and 2000 units / mL penicillin-streptomycin double antibody 2 The conditions were cultivated for 3 days, and then the cells were washed 3 times with serum-free DMEM culture medium to obtain cell carriers; the LMH cells were cultured in a microcarrier reactor, and the amount of microcarriers used was 8 grams per liter.

[0065] Inoculation of S2 virus: inoculate the avian influenza virus H9 subtype virus liquid into the cell carrier obtained in step S1 according to the final volume of 1:180, place it at 37°C for 60 minutes after adsorption, discard the virus liquid, then add medium A, in 37°C, 5% CO 2 72h under the condition of culture, 80% cytopathy occurs, and diseased cells are obtained;

[0066] Described m...

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Abstract

The invention belongs to the technical field of veterinary biological products and particularly relates to a preparation method of an avian influenza virus H9 subtype inactivated vaccine. An LMH passage cell line is adopted to serve as a carrier cell for viral multiplication, a culture medium prepared from glutamine, recombinant human insulin, human serum albumin, transferrin, biotin and a growth factor is adopted for culture, and virus liquid is collected, inactivated and prepared into the vaccine. The LMH passage cell line is adopted to perform avian influenza virus H9 subtype viral multiplication without additional pancreatin adding, and the LMH cell is clear in background, free of extraneous pathogens and easy to multiply. The process can be effectively simplified, and the cost is reduced. In addition, the avian influenza virus H9 subtype inactivated vaccine prepared by adopting the preparation method is high in virus content and good in stability and safety and is a more ideal avian influenza virus H9 subtype inactivated vaccine.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a preparation method of an avian influenza virus H9 subtype inactivated vaccine. Background technique [0002] H9N2 subtype avian influenza belongs to low pathogenicity avian influenza, but its incidence rate is high. Its main clinical manifestations are mild respiratory symptoms, reduced feed intake, egg production rate can drop from 90% to below 20%, or even stop production. About 30% of commercial broilers can die, and they are easily mixed with Escherichia coli, which seriously affects the production performance of poultry and brings serious economic losses to the poultry industry. Moreover, the H9N2 subtype AIV can pass through host barriers and infect mammals, including humans, and has the opportunity to spread in humans on a large scale, posing a serious threat to human health. [0003] Although the inactivated vaccine of H9 subtype avia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/145A61P31/16C12N7/00
CPCA61K39/12A61K2039/5252A61K2039/575C12N7/00C12N2760/16134C12N2760/16151
Inventor 张毓金谢秉超严悌昆敖艳华
Owner 广州渔跃生物技术有限公司
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