Attenuated strain of grass carp reovirus II and application of attenuated strain
A technology of reovirus and attenuated strains, applied in the directions of viruses, antiviral agents, viral antigen components, etc., can solve the problem that the protection power needs to be improved, etc.
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Embodiment 1
[0028] Isolation and cultivation of grass carp reovirus type II:
[0029]The applicant purchased healthy grass carp for the test from a farm in Sanshui District, Foshan City, Guangdong Province. When using conventional PCR methods to exclude whether the test fish carried GCRV pathogens, GRCVⅡ virus was detected from one of the fish (this virus is present in this country). Invention or referred to as GCRV II-GD1207). Take the liver, kidney and spleen of GCRV-positive fish, wash them twice with PBS (pH7.2), weigh them, cut the tissues into small pieces, grind them with a tissue homogenizer, and use M199 medium to make 1: 10 tissue homogenate, after repeated freezing and thawing for 3 times, it was centrifuged at 3000rpm / min, 6000rpm / min and 10000rpm / min in turn, each time for 10min to remove sediment, and the supernatant was filtered through a microporous filter with a filter membrane pore size of 0.22μm Sterilized spare. After the CIK cells grown into a dense monolayer were r...
Embodiment 2
[0040] The safety of GCRV II-GD1207 in the preparation of grass carp hemorrhagic disease live vaccine:
[0041] After the prepared CIK cell virus liquid was filtered and sterilized, it was diluted to a GCRV II-GD1207 virus titer of 80 copies / μL, and then injected with grass carp with an average body length of 10-15 cm (weight of 20-25 g) and an average body length of 4 ±0.5cm (body weight: 5±0.5g) rare gobi carp, and a negative control group injected with sterile PBS, each grass carp intraperitoneally injected with 0.2ml, and rare gobi carp with 0.05ml intraperitoneally injected, each group of test fish Both are 20 tails. During the test, the water temperature was controlled at about 28°C, and the disease and death of the test fish in each group were observed every day for 3 consecutive weeks. At the same time, during the experiment, RT-PCR was used to detect the GCRV virus in the fish that did not die or became ill after 2 weeks of challenge.
[0042] The results showed tha...
Embodiment 3
[0044] Application of GCRV II-GD1207 in the preparation of grass carp hemorrhagic disease live vaccine:
[0045] Healthy grass carp (average body length 10-15cm, body weight 20-25g) not carrying GCRV virus were randomly divided into 4 groups, injection immunization group, immersion immunization group, oral immunization group and control group, 60 fish in each group. Dilute the attenuated vaccine virus content to 80copies / μL, inject 200μL per tail in the immunized group, soak the final concentration of vaccine virus in the immunized group to 10copies / μL, add 1% NaCl to soak for 1 hour, and oral vaccine with 1×10 3 Copies / μL GCRV II-GD1207 virus solution is sprayed on the surface of conventional fish feed by spraying, spraying 100mL of virus solution per kg of feed, drying at room temperature for 1-2 hours, and then spraying with appropriate amount of cod liver oil and virus Mix the feed with liquid, so that the cod-liver oil is wrapped on the outermost surface of the feed, so a...
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