Expression and identification of p15 in BAC-TO-BAC baculovirus expression systems.

To characterize the properties and functions of chicken p15 protein,BAC-TO-BAC baculovirus expression systems was used to express active p15 protein.The multiple clone site of donor plasmid in this system was reconstructed.The donor plasmids of p15 fused TAP-tag for purification, pFAST-NMCS-N-TAP-p15 and pFAST-NMCS-C-TAP-p15,were constructed and transformed into DH10Bac competent cells,respectively.From two transformants, Bacmid-N-TAP-p15 and Bacmid-C-TAP-p15, were isolated after three times monoclonal plating. Two recombinant bacmid were extracted and transfected into sf9 cells and then the recombinant virus was harvested from cell culture supernatant at 72 hours after transfection.The titer assay was performed after amplifying the recombinant baculoviral stock.sf9 cells were infected with two recombinant baculovirus as high titer respectively for the expression of p15 fusion proteins.The results showed that the titers of N-TAP-p15 and C-TAP-p15 recombinant baculovirus were 3.6×107 pfu/mL and 5.4×107 pfu/mL,respectively.Two p15 fusion proteins were expressed in sf9 cells and amounting to 4.32% and 4.7%,respectively.Western blotting showed that both of them specifically bound to McAb against p15 and rabbit IgG,respectively.