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Tách dòng và biểu hiện gen mã hoá tiểu đơn vị P66 của enzyme phiên mã ngược của virus HIV – 1

Authors
Publisher
Vien Khoa Hoc Va Cong Nghe Viet Nam
Publication Date
Keywords
  • Cloning Vector
  • Expression Vector
  • Human Immunodeficiency Virus Type I
  • Reverse Transcriptase-Rt
  • Subunit P66
  • Subunit P51
  • Tách Dòng
  • Biểu Hiện Gen
  • Gen Mã Hoá
  • Enzyme Phiên Mã Ngược
  • Virus Hiv – 1
Disciplines
  • Biology

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

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