Duck reovirus, duck tembusu virus and duck adenovirus triple inactivated vaccine and preparation method thereof
A technology of duck Tembusu virus and reovirus, applied in biochemical equipment and methods, vaccines, viruses, etc., can solve problems such as mutual interference and reduced immune effect, achieve stable quality, simple preparation method, and strengthen duck The effect of the body's immune function
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Embodiment 1
[0051] S1. Preparation of cell carrier: After digestion and dispersion of LMH cells with 0.25% trypsin, DMEM / F12 medium with 5% newborn calf serum added to the cell monolayer at 37°C and 5% CO2 in a spinner flask , Discard the DMEM / F12 culture medium of 5% newborn calf serum, inoculate the cell maintenance medium, wash the cells with serum-free DMEM / F12 culture medium 3 times to obtain the cell carrier;
[0052] The cell maintenance fluid contains serum-free DMEM / F12 culture fluid, 2mM / ML glutamine, 0.2mM / ML N-N-diethyl-1-4-phenylenediamine;
[0053] S2. Virus inoculation: inoculate the viral liquids of duck reovirus, duck tambusu virus and duck adenovirus in a volume of 4:4:2 into the cell vector obtained in step S1, and place it at 37°C for adsorption Aspirate and discard the virus solution after 30 minutes, and then add DMEM / F12 culture solution containing 1% newborn calf serum and culture for 48 hours at 37°C and 5% CO2. Harvest the diseased cells when the cytopathic effect rea...
Embodiment 2
[0063] S1. Preparation of cell carrier: After digesting and dispersing LMH cells with 0.25% trypsin, adding 7% newborn calf serum to the DMEM / F12 culture solution in a spinner flask at 37°C and 5% CO2, after it is cultured to the cell monolayer , Discard the DMEM / F12 culture medium of 7% newborn calf serum, inoculate the cell maintenance medium, and wash the cells with serum-free DMEM / F12 culture medium for 3 times to obtain the cell carrier;
[0064] The cell maintenance fluid contains serum-free DMEM / F12 culture fluid, 2mM / ML glutamine, 0.2mM / ML N-N-diethyl-1-4-phenylenediamine;
[0065] S2. Virus inoculation: inoculate the viral liquids of duck reovirus, duck tambusu virus and duck adenovirus in a volume of 4:4:2 into the cell vector obtained in step S1, and place it at 37°C for adsorption Aspirate and discard the virus solution after 35 minutes, then add 1.5% newborn calf serum-containing DMEM / F12 culture solution and culture for 48 hours at 37°C and 5% CO2. When the cytopathic...
Embodiment 3
[0075] S1. Preparation of cell carrier: After digesting and dispersing LMH cells with 0.25% trypsin, adding 9% newborn calf serum in DMEM / F12 culture solution in a spinner flask at 37°C and 5% CO2, after culturing to the cell monolayer , Discard the DMEM / F12 culture medium with 9% newborn calf serum, inoculate the cell maintenance medium, wash the cells 3 times with serum-free DMEM / F12 medium to obtain the cell carrier;
[0076] The cell maintenance fluid contains serum-free DMEM / F12 culture fluid, 2mM / ML glutamine, 0.2mM / ML N-N-diethyl-1-4-phenylenediamine;
[0077] S2. Virus inoculation: inoculate the viral liquids of duck reovirus, duck tambusu virus and duck adenovirus in a volume of 4:4:2 into the cell vector obtained in step S1, and place it at 37°C for adsorption After 40 minutes, aspirate and discard the virus solution, then add DMEM / F12 culture solution containing 2% newborn calf serum and culture for 48 hours at 37°C and 5% CO2. When the cytopathic effect reaches 80%, har...
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