Attenuated strain YN150 of variant porcine epidemic diarrhea virus and applications thereof
A technology for porcine epidemic diarrhea and attenuated strains, applied in the direction of viruses, antiviral agents, and virus antigen components, can solve the problems of inability to provide protection and ineffective prevention of diarrhea, etc., achieve infection prevention, simple preparation process, and easy storage and transportation Effect
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Embodiment 1
[0050] The collection and processing of embodiment 1 disease material
[0051] The disease samples were collected from the small intestine of a 3-day-old piglet that was immunized with porcine transmissible gastroenteritis-porcine epidemic diarrhea inactivated vaccine in Yunnan Province but died of an outbreak of PED. The porcine epidemic diarrhea virus was detected by RT-PCR. Add physiological saline to the small intestine of piglets at a ratio of 1:9 (volume ratio), mash, freeze and thaw twice, centrifuge at 2000r / min, 4°C for 15min, collect the supernatant, and add 1500ml of penicillin and streptomycin per ml. Unit, placed in a 4°C refrigerator overnight, and then centrifuged at 10,000r / min, 4°C, 15min, the supernatant was taken, filtered with a 0.22μm filter, and stored in a -80°C refrigerator.
Embodiment 2
[0052] Isolation and identification of embodiment 2 virus
[0053] Virus isolation Take out the sample used as infected cells from the -80°C refrigerator, add trypsin to the solution at 50 μg / mL, put it in a 37°C incubator for 30 minutes, and finally distribute 1mL per tube into different 1.5mL Store in sterilized EP tubes for later use. Take the Vero monolayer cells in good growth condition, discard the culture medium, and wash the monolayer cells twice with PBS solution. According to 25cm 2 Add 1 mL of the treated disease material sample for infection into the culture bottle, and add trypsin at a final concentration of 10 μg / mL, and incubate at 37°C CO 2 Adsorb in the incubator for 1 hour, during which it was shaken once, and normal cells were set up as a control. Discard the adsorption solution (adsorption solution after the second generation can not be discarded), and add 5 mL of DMEM maintenance solution (serum-free) containing 10 μg / mL final concentration of trypsin, ...
Embodiment 3
[0058] Passage weakening of embodiment 3 virus
[0059]The 80% confluent Vero cells grown for 24 hours were washed twice with serum-free DMEM, and inoculated with 0.1 MOI of YN1 according to the method of detoxification above, and the maintenance solution was DMEM containing trypsin at a concentration of 8 μg / mL. When about 36 hours, when the cytopathic effect reached 85%, the virus was harvested, and the virus was stored in a refrigerator at -80°C. The collected viruses were repeatedly frozen and thawed three times and then serially passaged.
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