Biological preparation for enhancing resistance of pigs to viral epidemic diseases
A biological agent and viral technology, applied in the field of biology, can solve the problems of high production cost of interferon, the impact of IFN antiviral effect, limited clinical trials, etc., and achieve the effect of easy purification, high activity and high quality
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Embodiment 1
[0042] Example 1: pIFN-α, pIFN-γ and pIFN-λ1 gene sequence optimization and protein expression
[0043] In the early stage, we connected the pIFN-α, pIFN-γ and pIFN-λ1 genes to the eukaryotic expression system (insect cell baculovirus expression system), transfected Sf9 cells for expression, and selected Sf9 cells in good condition to inject 1×10 6 The cell / mL density was laid on a 6-well plate, and the DNA transfection was carried out when the cell density was about 70%. According to the instructions of X-tremeGENE HP DNA TransfectionReagent, each well was transfected with 2 μg of DNA, mixed well, at 37°C, 5% CO 2 Cultivate in an incubator for 24 hours, observe the state of the cells until the cells become larger. The supernatant was collected, and the obtained recombinant virus was blindly passed for 3 generations, and then the virus was collected. The obtained recombinant virus was used to infect sf9 cells, and verified by IFA and Western blot. The results showed that rec...
Embodiment 2
[0044] Example 2: pIFN-α, pIFN-γ, pIFN-λ1 stimulated antiviral gene expression detection
[0045]In order to verify the antiviral activity of the obtained pIFN-α, pIFN-γ, and pIFN-λ1, the expression of antiviral genes induced by them was detected first. Select PK-15 / CRL-2843 cells in good condition to 2.7×10 5 The cell / mL density was spread on a 24-well plate, and the cell density was equal to a monolayer for pIFN induction. Dilute pIFN, add 100 μl to each well, and store pIFN-α at 37°C, 5% CO 2 Cultivate in an incubator for 12 hours; pIFN-γ and pIFN-λ1 at 37°C, 5% CO 2 Cultivate in the incubator for 22h. The old cell culture medium was discarded, the cells were washed with pre-cooled PBS, the cells were lysed by adding RNAiso, and the cellular RNA was extracted and reverse-transcribed for quantitative PCR verification (see Table 1 for primers used in quantitative PCR).
[0046] The results of fluorescence quantification showed that in PK-15 cells, the dilution of pIFN-α w...
Embodiment 3
[0051] Example 3: The optimal inoculation dose of VSV / PRV infecting PK15 / CRL-2843 cells.
[0052] In the present invention, the antiviral effects of pIFN-α, pIFN-γ, and pIFN-λ1 are tested using the VSV system and porcine pseudorabies virus (PRV). Therefore, the priority is to detect the optimal inoculated dose of VSV and PRV infecting PK15 / CRL-2843 cells, and the selection standard is that 50% of the cells are infected as the optimal inoculated dose. Spread well-growing PK-15 cells and CRL-2843 cells in proportion on 24-well cell plates. After 12 hours, PK15 cells were inoculated with VSV / PRV at 1, 0.5, 0.1, and 0.01 MOI, and CRL-2843 cells were inoculated at 1 MOI. , 2, 3, 4, 5, 6, 7 MOI for inoculation, 12 hours after the virus infected the cells, take out the cell plate from the cell culture incubator, wash it twice with PBS, add trypsin to each well for digestion, and digest in the cell incubator for 5 min , add DMEM containing FBS, stop digestion, collect into 1.5mL EP t...
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