Use of an arrayed promoter-probe library for the identification of macrophage-regulated genes in Mycobacterium tuberculosis
Use of an arrayed promoter-probe library for the identification of macrophage-regulated genes in Mycobacterium tuberculosis
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DOI：
10.1099/00221287-148-5-1571
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发表时间：
2002-05-01
期刊：
影响因子：
2.8
通讯作者：
Dale, JW
中科院分区：
文献类型：
--
作者：
Hobson, RJ;McBride, AJA;Dale, JW
结核分枝杆菌在感染后在人体内的生存，特别是在巨噬细胞内，可能需要一些分枝杆菌基因的激活。为了鉴定这类基因，利用大肠杆菌-分枝杆菌穿梭载体，构建了结核分枝杆菌H37Rv DNA启动子-探针文库，并将其插入LacZ报告基因的上游。随后用该文库转化牛分枝杆菌卡介苗(BCG)，4800个BCG克隆以96孔微量滴定的形式排列，从而能够在各种条件下测试单个克隆的启动子活性。从初步筛选中，筛选出了41个在酸化亚硝酸钠作用下LacZ表达上调的克隆。随后的序列分析确定了其中26个克隆含有潜在的启动子。在检测了从THP-1巨噬细胞系回收的卡介苗克隆中LacZ的表达后，选择了三个基因，用实时荧光定量RT-PCR检测了它们在巨噬细胞感染期间结核分枝杆菌中的表达。其中两个基因，Rv126S(功能未知)和Rv2711(编码铁依赖的抑制蛋白)，显示出在巨噬细胞中上调的证据。
The survival of Mycobacterium tuberculosis within the human host after infection, especially within macrophages, is likely to require the activation of a number of mycobacterial genes. To identify such genes, a promoter-probe library was constructed in which fragments of M. tuberculosis H37Rv DNA were inserted upstream of a lacZ reporter gene, using an Escherichia coli-mycobacterial shuttle vector. Mycobacterium bovis Bacille Calmette-Gudrin (BCG) was subsequently transformed with this library and 4800 BCG clones were arrayed in a 96-well microtitre format, enabling the testing of individual clones for promoter activity under a variety of conditions. From preliminary screening, 41 clones were selected that exhibited upregulation of lacZ expression when subjected to acidified sodium nitrite. Subsequent sequence analyses identified 26 of these clones as containing potential promoters. After measuring lacZ expression in BCG clones recovered from a THP-1 macrophage cell line, three genes were selected for assessment of their expression in M. tuberculosis during macrophage infection, by realtime RT-PCR. Two of these genes, Rv126S (with unknown function) and Rv2711 (encoding the iron-dependent repressor protein Well), showed evidence of being upregulated within macrophages.