Recombinant virus of chimeric IBV H120 S1 gene ectodomain suitable for cell culture and construction method and application thereof
An IBVH120S1, cell culture technology, applied in microorganism-based methods, medical raw materials derived from viruses/phages, antiviral agents, etc., can solve the problems of exogenous virus contamination cost, small toxin production, and long production cycle.
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Embodiment 1
[0053] Embodiment 1 Construction of IBV recombinant virus BeauR-H120 (S1) of the present invention
[0054] The specific construction method is completed by the following steps:
[0055] 1. Total RNA was extracted from IBV H120 vaccine strain and Beaudette P65 generation cytotoxicity with Trizol reagent (Invitrogen, USA). The downstream primers in Table 1 were used for reverse transcription, wherein primer Beau-H21711R was used for reverse transcription of the extramembrane region of S1 gene of IBV H120 strain. Primers Beau-5752R, Beau-8693R, Beau-15520R, Beau-20422R, and Beau-27608R were used for reverse transcription of the backbone fragment of the parent virus Beaudette strain.
[0056] The primer pair used when the recombinant virus BeauR-H120 (S1) was constructed in table 1
[0057]
[0058] 2. Use the primer pair Beau-H20421F and Beau-H21711R in Table 1 to amplify the extramembrane region fragment of the S1 gene of IBV H120 strain, and the resulting fragment is labe...
Embodiment 2
[0073] Embodiment 2 The cultivation of IBV recombinant virus BeauR-H120 (S1) of the present invention
[0074] 1 method
[0075] 1.1 Stable passage of rescued successful IBV recombinant virus
[0076] The successfully rescued recombinant virus was serially passaged on Vero cells. When the Vero cells just covered the cell bottle, inoculate 1 mL of the recombinant virus solution that was repeatedly frozen and thawed 3 times, and add 4 mL of serum-free DMEM cell maintenance solution, and place at 37 ° C, 5% CO 2 Continue culturing in the cell culture incubator. The virus was harvested when 90% of the cells showed cytopathic changes, and the entire cell bottle was placed in a -70°C refrigerator for three times of repeated freezing and thawing, waiting for the next inoculation. 1mL cytotoxic RNA was extracted for each generation, and the presence of IBV genome was detected by RT-PCR.
[0077] 1.2 Determination of virus titer
[0078] Take the 5th, 10th, 15th, 18th, and 21st ge...
Embodiment 3IB
[0100] Immune efficacy evaluation of embodiment 3 IBV recombinant virus BeauR-H120 (S1)
[0101] 1 method
[0102] 1.1 IBV recombinant virus BeauR-H120 (S1) immune protection test
[0103] Randomly divide 40 1-day-old SPF chicks into A and B groups (20 / group), and raise them in negative pressure isolators respectively. At the age of 7 days, group A was inoculated with IBV BeauR-H120 (S1) cytotoxicity (21st generation) (10 6.25 EID 50 / 0.1mL), each eye drops 0.1mL. Group B was used as the control group, and 0.1 mL of normal Vero cell lysate was instilled in each eye. From the date of inoculation, observe and record the incidence of the vaccinated chickens every day, and 14 days and 21 days after immunization, blood was collected from the two groups of chickens, and the specific antibody was detected after the serum was separated. The specific method was carried out according to the instructions of the IBV antibody detection kit of IDEXX company in the United States. 21 da...
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