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
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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
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