Duck BYD virus inactivated vaccine and preparation method thereof
A technology for inactivating vaccines and viruses, applied in biochemical equipment and methods, antiviral agents, viruses/phages, etc., can solve the problems of lack of effective treatment and immune preventive measures, lack of effective treatment drugs for flavivirus infection, etc. Achieve excellent immunogenicity, good immune effect and good safety effect
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Embodiment 1
[0028] Example 1 Virus isolation and pathogenicity
[0029] 1. Isolation and identification The virus was isolated from the brain tissue of infected ducks in a duck farm in Baiyangdian, Hebei Province, China. Make 10% homogenate of diseased duck brain tissue samples with sterile PBS, centrifuge at 3000r / min for 20min, filter and sterilize, inoculate 9-12 days old duck embryos without maternal antibody through allantoic cavity, and incubate at 37°C for 5 On the first day, the allantoic fluid was harvested and passaged in duck embryos. Inoculate the second-generation allantoic fluid poison on the BHK-21 cell monolayer, culture it statically in a 5% carbon dioxide incubator, and observe it for 7 days. When the cell changes appear, the culture is harvested and stored at -70°C.
[0030] Prepare the ultrathin sections of infected cells and observe under the transmission electron microscope. A large number of spherical enveloped virus particles with a diameter of about 50 nm can be ...
Embodiment 2
[0034] Example 2 Preparation of duck BYD virus JXSP strain virus solution
[0035] 1. Propagation of virus seeds Dilute BYDV-JXSP 2nd generation allantoic duck embryo venom 5 times and inoculate it into BHK-21 cell culture, place it in a 5% carbon dioxide incubator at 37°C for static culture until 75% of the cells have lesions Harvest the cell fluid, freeze and thaw twice, take the supernatant and dilute it 100 times, then infect and inoculate BHK-21 cells, pass it to the 8th passage, harvest the cell fluid and store it at -70°C as virus seeds, the virus content is ≥10 5.5 TCID 50 / mL.
[0036] 2. Cell seed propagation Take the BHK-21 cells stored in liquid nitrogen, immediately place them in a 37°C water bath to melt, centrifuge at 800r / min for 5min, discard the supernatant, add DMEM cell growth medium containing 10% fetal bovine serum to suspend the cell pellet, Transferred to a cell culture bottle, cultured statically at 37°C in a 5% carbon dioxide incubator, and subcultu...
Embodiment 3
[0038] Example 3 Preparation of duck BYD virus inactivated vaccine
[0039] 1. Inactivation of virus fluid
[0040] Add the virus solution prepared in Example 2 to the formaldehyde solution at a final concentration of 0.2% (volume ratio), mix well, shake well, and then act at 37° C. for 24 hours to inactivate the virus.
[0041] 2. Inactivation effect test
[0042] BHK-21 cells were cultured to a monolayer on a 24-well plate, and the 10-fold diluted BYDV-JXSP virus solution treated with formaldehyde inactivation in step 1 was inoculated on the BHK-21 cell monolayer at 0.2 mL / well, and each sample Inoculate 3 wells, discard the sample solution after incubation for 1 hour, continue to maintain the culture after washing with maintenance solution, and set positive control and blank control wells at the same time; observe the positive control wells after 48 hours. Cytopathies can be seen, and the samples and blank control wells are negative; Blind passage for 2 generations, the s...
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