Method for constructing recombinant virus for expressing ALV-K envelope protein
A technology of ALV-K and envelope protein, applied in the direction of retroRNA virus, virus, viral peptide, etc., can solve the problem of low expression level of viral protein, no commercial specific ALV-K antigen and antibody detection kit, The detection effect is not very good and other problems, to achieve the effect of speeding up the purification process
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[0021] (1) Preparation of ALV-K proviral DNA:
[0022] Take 400uL of the supernatant after ALV-K infection of the cells in a 1.5mL finger tube, add 400uL of cell lysate (50mmol / L Tris-HCl pH=8.0, 20mmol / L EDTA pH=8.0, 2% SDS and proteinase K), fully After mixing, place it in a 56°C water bath for 4 hours; add 400uL Tris to balance the phenol, mix well and centrifuge at 10000rpm for 10min, take the supernatant into another 1.5mL finger tube; add 400uL phenol: chloroform: isoamyl alcohol, and mix well Then centrifuge at 10,000rpm for 5min, take the supernatant into another 1.5mL finger tube; add 800uL of absolute ethanol, invert and mix well, incubate at -20°C for 30 minutes, centrifuge at 12,000rpm for 15 minutes, discard the supernatant; after natural drying at room temperature Add 30uL sterilized ultrapure water and 2uL RNase to the precipitate, fully dissolve to obtain ALV-K proviral DNA, and store at -20°C for later use.
[0023] (2) PCR amplification of ALV-K env gene fra...
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