A method for producing porcine pseudorabies virus antigen by whole suspension cell culture
A porcine pseudorabies virus and cell culture technology, applied in the directions of virus antigen components, biochemical equipment and methods, viruses, etc., can solve the problems of producing porcine pseudorabies virus antigens, difficult to achieve expansion culture, low antigen titers, etc. Conducive to cell absorption, improved product quality, and easy to expand the effect of culture
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Embodiment 1
[0035] Embodiment 1 Keratan sulfate oligosaccharide preparation and component analysis
[0036]
[0037] Preparation of Group A:
[0038] Take 50g of keratan sulfate, dissolve it in 300ml of 0.1M acetate buffer (pH6.0), add 25U of mixed enzyme, and degrade at 37°C for 24h. After the reaction, 2 times the volume of ethanol was added, stirred, left overnight at room temperature, centrifuged at 4000 rpm for 15 min, and the supernatant (supernatant A) was taken. Add 300ml of distilled water to the precipitate to dissolve, add 3 times the amount of ethanol, stir, leave at room temperature overnight, centrifuge at 4000rpm for 15min, and take the supernatant (supernatant B). Mix supernatant A and supernatant B, concentrate under reduced pressure, use a Bio-Gel-P-2 column (3.6╳134cm), use distilled water as a solvent, perform gel filtration, and freeze-dry the filtrate.
[0039] The preparation of keratan sulfate oligosaccharides in groups B-D refers to group A.
[0040] Composi...
Embodiment 2
[0044] The influence of embodiment 2 keratan sulfate oligosaccharides on BHK-21 cell growth
[0045] BHK21 cells were cultured with keratan sulfate oligosaccharides prepared in Example 1A-D groups, specifically:
[0046] Take the BHK21 cells out of the liquid nitrogen tank and add them to the DMEM medium containing 10% newborn calf serum, at 37°C, 5% CO 2 cultured until it grows into a good monolayer, then digested with an appropriate amount of trypsin digestion solution containing 0.02% EDTA, digested at 37°C for 6 min, and adjusted the cell density to 5×10 with DMEM medium containing 10% newborn bovine serum. 5 cells / mL of cell suspension, inoculate in shake flasks, add nutrient solution containing 2% newborn bovine serum at 37°C, 5% CO 2 The culture was carried out in the incubator, and the rotation speed was set to 150rpm / min for suspension culture until the cell density reached 4×10 6 each / mL, wherein, the nutrient solution containing 2% neonatal bovine serum contains 1...
Embodiment 3
[0050] Example 3 Production of Porcine Pseudorabies Virus Antigen by Full Suspension Cell Culture
[0051] (1) Resuscitate the seed cell BHK21, and use a shaker flask for suspension culture expansion: take the BHK21 cell species out of the liquid nitrogen tank for resuscitation, add it to DMEM medium containing 10% newborn calf serum, and store it at 37°C, 5% CO 2 cultured until it grows into a good monolayer, then digested with an appropriate amount of trypsin digestion solution containing 0.02% EDTA, digested at 37°C for 6 min, and adjusted the cell density to 5×10 with DMEM medium containing 10% newborn bovine serum. 5 cells / mL of cell suspension, inoculated in shake flasks, added nutrient solution containing 3% newborn bovine serum at 37°C, 5% CO 2 The culture was carried out in the incubator, and the rotation speed was set to 150rpm / min for suspension culture until the cell density reached 4×10 6 each / mL, wherein, the nutrient solution containing 3% neonatal bovine serum...
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