Expression, purification and bioactivity test of human immunodeficiency virus P66 protein
The purpose of this study is to express and purify the HIV-1 P66 subunit of transcriptase in vitro and to obtain the pure bioactive recombinant P66 protein.The full length HIV p66 gene fragment of HIV-1 HXB2 was amplified by PCR and inserted into pTWIN1 vector to generate p66 expression plasmid pTWIN-p66.After transforming pTWIN-p66 into E.coli(BL21),the expression of p66 gene was induced by IPTG.The expressed purified P66 protein was prepared in one step by chitin pre-packed column.In order to compare the bioactivity between purified P66 protein and M-MLV RT in kit, the total RNA of B95-8 cell and the control RNA in RT-PCR kit were catalyzed by purified P66 protein and M-MLV RT respectively under identical conditions, then the products of RT-PCR were electrophoresed on agarose gel. The result indicated that a high-level expression of HIV P66 protein could be induced by IPTG in bacterial system using expression plasmid pTWIN-p66. The final P66 protein by one step purification could reach 88%. The purified P66 had good bioactivity invitro, that was similar to the test of M-MLV RT. This study lays a foundation for developing domestic HIV confirmation test kit and for anti-HIV drug.