用于 cDNA 合成的人免疫缺陷病毒 1 型逆转录酶的酶学特征。

Enzymatic characterization of human immunodeficiency virus type 1 reverse transcriptase for use in cDNA synthesis.

机构信息

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

出版信息

Appl Biochem Biotechnol. 2013 Jan;169(1):77-87. doi: 10.1007/s12010-012-9953-8. Epub 2012 Nov 13.

DOI:10.1007/s12010-012-9953-8

PMID:23149716

Abstract

The aim of this study is to explore the advantages of using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in cDNA synthesis. Recombinant HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT were produced in an Escherichia coli expression system. In the incorporation of dTTP into poly(rA)-p(dT)(15) (T/P), the K (m) values for dTTP of HIV-1 M RT and HIV-1 O RT were 8 and 12 % of that of Moloney murine leukemia virus (MMLV) RT, respectively, and the K (m) values for T/P were 25 and 23 % of that of MMLV RT, respectively. Compared with MMLV RT, HIV-1 M RT and HIV-1 O RT were less susceptible to formamide, which is frequently used for cDNA synthesis with a G + C-rich RNA to improve specificity. The high substrate affinity and low susceptibility to formamide of HIV-1 RT might be advantageous for its use in cDNA synthesis.

摘要

本研究旨在探讨使用人类免疫缺陷病毒 1 型（HIV-1）逆转录酶（RT）进行 cDNA 合成的优势。重组 HIV-1 M 型（HIV-1 M）RT 和 HIV-1 O 型（HIV-1 O）RT 在大肠杆菌表达系统中产生。在将 dTTP 掺入 poly(rA)-p(dT)(15)（T/P）中时，HIV-1 M RT 和 HIV-1 O RT 的 dTTP 的 K（m）值分别为 Moloney 鼠白血病病毒（MMLV）RT 的 8%和 12%，而 T/P 的 K（m）值分别为 MMLV RT 的 25%和 23%。与 MMLV RT 相比，HIV-1 M RT 和 HIV-1 O RT 对常用于与富含 G+C 的 RNA 进行 cDNA 合成以提高特异性的甲酰胺的敏感性较低。HIV-1 RT 的高底物亲和力和对甲酰胺的低敏感性可能有利于其在 cDNA 合成中的应用。