Grass carp reovirus VP35 protein subunit vaccine, preparation method and applications thereof
A technology of subunit vaccine and reovirus, which is applied in the field of genetic engineering and molecular immunology, can solve the problem of preparing subunit vaccine from grass carp reovirus VP35 protein, achieve good immune protection effect, and the preparation method is simple , the effect of clear ingredients
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Embodiment 1
[0027] Construction of Grass Carp Reovirus V35 Protein Subunit Vaccine Expression Vector
[0028] Grass carp reovirus was isolated from a breeding pond in Xinxiang City, Henan Province. Grass carp viscera suffering from grass carp hemorrhagic disease were obtained, mixed with PBS buffer, ground and homogenized, and the homogenate was centrifuged at 8000g, and the supernatant was filtered through a filter with a pore size of 0.22µm , the filtrate was inoculated into the grass carp kidney cells (CIK) cultured in vitro, and the cells were collected after one week of culture, and the cells contained the proliferating grass carp reovirus. RT-PCR detection showed that the grass carp reovirus was type II grass carp reovirus. Using the isolated grass carp reovirus genome dsRNA as a template, the reverse transcription was divided into two steps. The reverse transcription system was as follows: 5 µL of total RNA was extracted, 1 µL of random primers, RNase free dH 2 O 4µL, the reaction...
Embodiment 2
[0031] Preparation of Grass Carp Reovirus VP35 Protein Subunit Vaccine
[0032] (1) Bacterial liquid expansion culture and induced expression: Inoculate 10 µL of positive bacterial liquid containing VP35-pET32a into 3 mL of Amp-containing LB medium, and culture overnight at 37°C with shaking. Inoculate 3mL of the activated bacterial liquid into 300mL of new LB medium containing Amp for expanded cultivation, and incubate at 37°C and 200rpm for 4h. Take 3mL bacterial liquid to detect OD 600 to 0.6. After adding a final concentration of 0.5mM or 1mM IPTG to induce 2, 4, and 6h, the induced expression was detected by SDS-PAGE, and the final induction concentration was determined. The results showed that 1mM IPTG induced the maximum protein expression at 6h ( figure 2 ). Afterwards, 300 µL of IPTG was added to 300 mL of the bacterial solution, and the shaking culture was continued at 37°C for 6 h.
[0033] (2) Protein purification: collect the cells by centrifugation at 8000rp...
Embodiment 3
[0037] Application of Grass Carp Reovirus Subunit Vaccine
[0038] (1) Detect the vaccine protein concentration, and dilute it to 0.3-0.4 mg / mL with PBS buffer to obtain the vaccine protein.
[0039] (2) Grass carp fingerlings of the same size were selected with a body length of 10-12cm and a weight of 15-20g. The same batch of grass carp fingerlings were reared in a water temperature of 27°C with oxygen for two weeks before the experiment. They were randomly divided into 2 groups, 50 grass carp in each group, and continued to be raised temporarily for 1 week. The water temperature was controlled within the range of 27°C, aerated and oxygenated, and fed regularly once a day. Group 1 was the control group, which received 100 µL PBS buffer solution intraperitoneally; group 2 was the experimental group, which received 100 µL vaccine protein intraperitoneally.
[0040] (3) VP35 protein increases the number of immune cells: 1, 3, 5, 7, 14, 21, 28, and 35 days after the injection i...
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