CLONING AND EXPRESSION OF GENE ENCODING SUBUNIT P66 OF HIV-l REVERSE TRANSCRIPTASE

  • Phan Trọng Hoàng

Abstract

The human immunodeficiency virus type 1 (HIV-I) reverse transcriptase (RT) is a heterodimer comprised of two structurally distinct subunits (pSI and p66), RT is translated and assembled into virions as part of a precursor Gag-Pol polyprotein (PrI60Gag-Pol). Proteolytic processing of Prl60Gag-Pol by the pol-encoded protease (PR) generates the heterodimeric RT enzyme (p51/p66). The p66 subunit contains the DNA polymerase and RNase H domains. To elXpress subunit p66 in E. coli without using HIV the proteolytic processing protease, we isolated 1680 bp p66 coding region with specific primers: HIV P66-Ndel-F, HIV RT66-Sal1-R. This fragment was cloned into pCR2.1 vector and sequenced. P66 coding gene is inserted into pET2Ia(+) (Novagen) and expressed in rosetta-gamiTM strain (DE3) codon plus RP under inducing of (PTG. Optimized overexpression of subunit HIVI RT-p66 in E. coli is that final concentration ofIPTG was 1 mM and incubation was continued at 30°C for Sh

Tác giả

Phan Trọng Hoàng
điểm /   đánh giá
Published
2011-11-30
Section
Articles