Method for producing avian influenza virus bivalent inactivated vaccine by use of MDCK cell line

A bivalent inactivated vaccine and avian influenza virus technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, antiviral agents, etc., can solve difficult large-scale vaccine production, can not meet the needs of prevention and control, production process To achieve the effect of shortening the R&D cycle and production cycle, maintaining the stability of virus antigens, and facilitating large-scale production

Inactive Publication Date: 2014-01-29
乾元浩生物股份有限公司
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  • Application Information

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Problems solved by technology

This culture method is to propagate the virus by inoculating the virus into the allantoic cavity of chicken embryos. This production method cannot meet the needs of prevention and control of avian influenza in my country in the future, mainly in the following aspects: (1) Production cycle (2) limited by raw and auxiliary materials such as chicken embryos; (3) backward production technology, labor-intensive, and high production costs

Method used

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  • Method for producing avian influenza virus bivalent inactivated vaccine by use of MDCK cell line
  • Method for producing avian influenza virus bivalent inactivated vaccine by use of MDCK cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The planar adherent culture of embodiment 1 recombinant avian influenza virus H5N1 subtype Re-4 strain

[0024] Include the following steps:

[0025] (1) Use 850cm 2 MDCK cells were cultured in spinner flasks, and monolayer MDCK cells with good cell morphology were selected as cells for virus culture.

[0026] (2) Wash MDCK cells 3 times with 0.01mol / L, pH7.2-7.4 PBS.

[0027] (3) Dilute the recombinant avian influenza virus H5N1 subtype Re-4 strain with virus growth solution (DMEM solution containing 5 μg / mL TPCK-treated trypsin, 0.2% bovine serum albumin fraction V, pH 7.2-7.4) To contain virus MOI10 -4 -10 -6 .

[0028] (4) Inoculate the diluted virus solution into the MDCK cells washed in step (2), and place at 34°C, 5% CO 2 Cultivate in medium for 2-4 days, measure the titer of virus HA, add 0.1% formaldehyde solution to inactivate at 37°C for 24 hours, and harvest the virus antigen.

Embodiment 2

[0029] The bioreactor suspension culture of embodiment 2 recombinant avian influenza virus H5N1 subtype Re-4 strain

[0030] Include the following steps:

[0031] (1) Use TideCell-010 tidal high-density cell culture system for MDCK cell perfusion suspension culture:

[0032] TideCell-010 tidal high-density cell culture system was inoculated with 10000mL cells of appropriate concentration (1×10 6 -4×10 6 cells / mL). Connect the cell culture bottle to a culture bag (liquid tank) containing 40,000 mL of cell growth fluid for culture. The cell growth medium is 95% by volume of DMEM solution and 5% by volume of fetal calf serum, with a pH value of 7.2-7.4. By adjusting the culture conditions in the cell culture system: dissolved oxygen is 50%-100%, CO 2 The concentration is 5%, the glucose content is 500-4500mg / L, the pH value is 7.2-7.4, and the cell culture temperature is 37°C to cultivate high-density MDCK cells. Cultivate until the second day, and perform perfusion culture...

Embodiment 3

[0036] Embodiment 3 Planar adherent culture of recombinant avian influenza virus H5N1 subtype Re-6 strain

[0037] Include the following steps:

[0038] (1) Use 850cm 2 MDCK cells were cultured in spinner flasks, and monolayer MDCK cells with good cell morphology were selected as cells for virus culture.

[0039] (2) Wash MDCK cells 3 times with 0.01mol / L, pH7.2-7.4 PBS.

[0040] (3) Dilute the recombinant avian influenza virus H5N1 subtype Re-6 strain with virus growth solution (DMEM solution containing 5 μg / mL TPCK-treated trypsin, 0.2% bovine serum albumin fraction V, pH 7.2-7.4) To contain virus MOI10 -4 -10 -6 .

[0041] (4) Inoculate the diluted virus solution into the MDCK cells washed in step (2), and place at 34°C, 5% CO 2 Cultivate in medium for 2-4 days, measure the titer of virus HA, add 0.1% formaldehyde solution to inactivate at 37°C for 24 hours, and harvest the virus antigen.

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Abstract

The invention provides a method for producing avian influenza virus (H5 subtype and H9 subtype) bivalent inactivated vaccine by use of a MDCK cell line. The method comprises the following steps: (1) passage and culture of the MDCK cell line; (2) virus inoculation and reproduction; (3) inactivation and harvesting of virus liquid; and (4) preparation of the vaccine. The method for producing avian influenza virus inactivated vaccine by use of the MDCK cell line has the advantages that exogenous factor pollution is avoided, large-scale production is easy to realize, the virus antigen stability can be maintained well and the like; and moreover, the development period and production period of the vaccine can be shortened, and the ability of emergency supply of vaccine in outburst epidemic situation is realized.

Description

technical field [0001] The invention relates to the technical field of biological products, in particular to a method for producing a bivalent inactivated vaccine of avian influenza virus (subtype H5 and subtype H9) using MDCK cell lines. Background technique [0002] At present, the way of producing the inactivated vaccine of avian influenza virus in my country is mainly to use 9-11 days old healthy chicken embryos for cultivation. This culture method is to propagate the virus by inoculating the virus into the allantoic cavity of chicken embryos. This production method cannot meet the needs of prevention and control of avian influenza in my country in the future, mainly in the following aspects: (1) Production cycle (2) It is limited by raw and auxiliary materials such as chicken embryos; (3) The production process is backward, labor-intensive, and production costs are high. The avian influenza virus (H5 subtype + H9 subtype) bivalent inactivated vaccine plays a pivotal rol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/295C12N7/00A61P31/16C12R1/93
Inventor 王贺民黄文强周蕾蕾张翼陈秋阁严石李浩鹏李爱君万桂清
Owner 乾元浩生物股份有限公司
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