A kind of Newcastle disease virus recombinant vaccine strain of pigeon origin and its construction method and application
A technology of Newcastle disease virus and epidemic virus, applied in the direction of viruses, vaccines, applications, etc., can solve the problem of lack of pigeon Newcastle disease vaccine products, etc.
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Embodiment 1
[0043] Example 1: Rescue of LaSota strain
[0044] 1. Virus purification
[0045] The purification of the virus is to obtain a single virus clone. The LaSota strain was purified by the limiting dilution method. The detailed steps are as follows: Dilute the virus liquid by 10 times. SPF chicken embryos (100 μL / piece) were inoculated with 5 pieces per dilution. After 4 days, the chicken embryo allantoic fluid was harvested to measure the activity of HA. The allantoic fluid with the highest dilution factor with HA activity was selected as the next-generation purified virus fluid. The same method was used for doubling dilution and chicken embryo inoculation, and the virus was continuously purified for 5 generations, and the 5th generation virus liquid was stored in aliquots, which was used as the original seed virus for the next test.
[0046] 2. Virus sequence determination
[0047] Using the purified viral reverse-transcribed genomic cDNA as a template, 13 pairs of primers d...
Embodiment 2
[0068] Example 2 Rescue of vaccine candidate seed virus for recombinant pigeons
[0069] 1. Virus screening and purification
[0070] The pigeon-derived Newcastle disease virus of the VI.2.1.2.2 subtype isolated and preserved in the laboratory was selected for reproduction and rejuvenation, and then 5 strains were purified by the limiting dilution method. The hemagglutination titers are shown in Table 5. Finally, the QH1344 / 2017 strain after 5 generations of purification was selected as the original seed virus for the construction of full-length cDNA clones.
[0071] Table 5: Hemagglutination titer table of different strains of pigeon-derived Newcastle disease virus at each generation
[0072]
[0073] 2. Determination and analysis of the full-length genome sequence of QH1344 / 2017 strain
[0074]Using the purified QH1344 / 2017 virus reverse transcription genome cDNA as a template, using 13 pairs of primers designed to amplify the full-length sequence of type VI NDV (Table...
Embodiment 3
[0093] Example 3 Safety test and immune effect test of rLa-VI-QH17 vaccine strain on non-immunized pigeons
[0094] 1. Preparation of inactivated vaccine
[0095] The rLa-VI-QH17 vaccine strain was diluted 10 000 times with sterilized normal saline, inoculated with 9-11-day-old SPF chicken embryos, 0.1 mL per embryo, and incubated at 37°C. The dead embryos within 24h after inoculation were discarded, and the dead embryos were kept at 4°C in time for 24h~120h, and the mixed samples were collected. 10.5 , the virus titer is 10 9.48 EID 50 / 0.1mL. The Newcastle disease virus solution with the determined titer was introduced into the inactivation tank, and the final concentration of 0.1% formaldehyde solution was added to make it fully mixed, inactivated at 4°C for 48 hours, and shaken every 2 hours during the period. Oil adjuvant inactivated vaccine is prepared from the inactivated virus stock solution according to conventional methods. The same dose of LaSota inactivated va...
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