Bivalent attenuated vaccine for porcine epidemic diarrhea and porcine delta coronavirus and a preparation method thereof
A technology for porcine epidemic diarrhea and coronavirus, applied in biochemical equipment and methods, vaccines, viruses, etc., can solve the problems that there is no dual attenuated vaccine, and achieve non-toxicity, high safety, and good stability Effect
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[0034]Preparation of virus liquid for seedling production
[0035] Including cell expansion culture, virus inoculation, virus liquid harvest, virus titer determination, sterility and exogenous detection.
[0036] 1. Cell expansion in seed tanks: the density of suspended ST cells in shake flasks reaches 7×10 6 ~10×10 6 cells / mL and the viable cell rate is above 95%, transfer the cells to the seed tank. Before the transfer, calibrate the dissolved oxygen (DO) electrode, pH electrode, and temperature electrode of the seed tank, and the tank body is autoclaved. According to the volume of the seed tank, 20% of the total volume of the culture solution was pumped in at a rate of 0.5×10 6 ~1×10 6 The suspended ST cells were inoculated at a density of one / mL, and the culture conditions were 37° C., pH 7.2, DO 50%, and rotation speed 75 rpm.
[0037] 2. Virus inoculation: when the density of suspended ST cells in the bioreactor reaches 7×10 6 ~10×10 6 cells / mL, dilute the cell de...
Embodiment 1
[0043] The preparation method of embodiment 1 porcine epidemic diarrhea, porcine delta coronavirus dual attenuated vaccine
[0044] 1. Seed poison reproduction
[0045] For the adherent ST cells that grow well and cover a single layer, discard the original culture medium, and inoculate the porcine epidemic diarrhea virus cells according to the volume ratio of 1% of the original culture medium for passage to attenuate or porcine delta coronavirus cells To attenuate the toxicity, after 2 hours of adsorption, replace with DMEM maintenance solution containing 100 IU / mL each of penicillin and streptomycin, add trypsin with a final concentration of 10ug / mL, and store at 37°C, 5% CO 2 Cultivate in a concentration incubator for 24 to 72 hours, harvest the virus when the cytopathic rate reaches more than 80%, freeze and thaw twice in the refrigerator at -80°C, and then conduct sterility testing, mycoplasma testing, and exogenous virus testing to meet the requirements of the Veterinary ...
Embodiment 2
[0053] The virulence return strong test of embodiment 2 attenuated virus strains
[0054] Test method: each 2mL of the attenuated virus strain and the attenuated virus strain of porcine delta coronavirus cell passage (10 6 TCID 50 / mL) orally inoculated 3-day-old artificially raised piglets that did not eat colostrum, 5 piglets in the first group were orally administered 2 mL respectively, and they were killed 72 hours after planting to observe whether there were any abnormalities in the gastrointestinal tract tissue and prepare small intestine tissue filtrate (Mixed solutions of each individual), take 2 mL of small intestine tissue fluid orally inoculate the 3-day-old piglets of the second group, and kill them after 72 hours, observe and inoculate the piglets of the third group with the same method as above, and so on, and inoculate continuously for 5 generations (a total of 5 generations) Group).
[0055] Test results: The 3-day-old piglets were continuously subcultured fo...
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