Cell adaptation strain MJ of QX type IBV (Infectious Bronchitis Virus) and application of cell adaptation strain MJ
A technology for adapting strains and cells, applied in the field of chicken infectious bronchitis virus, can solve the problems of lack of IBV, lack of Vero cell adaptation strains, hidden dangers in biological safety, etc.
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Embodiment 1
[0037] The isolation and identification of embodiment 1 chicken infectious bronchitis virus
[0038] 1 Virus isolation
[0039] The disease material came from a large-scale farm in Jiangsu Province. Kidney tissues of dead chickens were aseptically collected, chopped and ground, and sterilized saline with double antibody content of 1000IU / mL was added at a ratio of 1:5 (v / v), 5000r / min Centrifuge for 5 minutes, take the supernatant and inoculate four 11-day-old SPF chicken embryos through the allantoic cavity, 0.2 mL / piece, and incubate at 37°C for 48 hours; discard the dead chicken embryos within 24 hours, and collect the allantoic fluid of 24-48 hours chicken embryos as The subcultured virus was named as IBYZ isolate and stored at -20°C.
[0040] 2 Identification of the virus
[0041] 1) Dwarfing test of chick embryos
[0042] After diluting the isolated IBYZ isolate strain 1:100 times with PBS, inoculate into the allantoic cavity of 5 11-day-old SPF chicken embryos, 0.2mL...
Embodiment 2
[0047] Embodiment 2 The cultivation of QX type chicken infectious bronchitis virus Vero cell adaptation strain MJ strain
[0048] 1 Continuous subculture of chick embryos isolated from IBYZ
[0049] Inoculate the allantoin of IBYZ isolates into the allantoic cavity of 4 SPF chicken embryos aged 9-11 days for subculture, 0.2mL / piece, and incubate at 37°C for 48 hours; discard the dead chicken embryos within 24 hours, and collect them for 24-48 hours Chicken embryo allantoic fluid was used as the subcultured virus. The harvested allantoic fluid was inoculated into chicken embryos again, and the above-mentioned process was repeated for 60 to 100 generations of continuous passage.
[0050] 2 Passage adaptation of Vero cells
[0051] The harvested allantoic venom of the 60th generation of IBYZ isolates was inoculated with a single layer of Vero cells, and placed in 37°C CO 2 After culturing in the incubator for 72 hours, freeze and thaw three times in the refrigerator, harvest t...
Embodiment 3
[0064] Example 3 Safety evaluation test of MJ strain
[0065] 1. Pathogenicity test on 3-day-old SPF chicks
[0066] The dosage is 10 6.5 The MJ strain cytotoxicity of TCID50 was used to inoculate 10 3-day-old SPF chicks through nasal drops and eye drops, and a PBS group was set up at the same time with a dose of 10 6.83 EID 50 The IBYZ virulent group was used as negative and positive controls. The results show that the chicks inoculated with IBYZ group began to die on the 4th day, and the mortality rate reached 60% during the test, while the PBS group and the MJ group did not observe obvious clinical symptoms during the whole test period, indicating that the MJ strain is safe and reliable for SPF chicks (Table 2).
[0067] Table 2 Pathogenicity test on SPF chicks
[0068]
[0069] 2. Virulence recovery test
[0070] The dosage is 10 6.5 TCID 50 Inoculate the first batch of 5 7-day-old SPF chicks through nasal drops and eye drops, and 7 days later, put the second ba...
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