结核分枝杆菌鼠李糖基转移酶基因(wbbL)的克隆和表达

Cloning and expression of M. tuberculosis wbbL gene enconding rhamnosyl transferase

关键词:  结核分枝杆菌  鼠李糖基转移酶  wbbL基因  基因克隆  基因表达

Key words:  M.tuberculosis  rhamnosyl transferase  wbbl gene  gene clone  gene expression

摘要:

[目的 ]从结核分枝杆菌基因组DNA中克隆鼠李糖基转移酶基因 (rhamnosyltransferase ,wbbL) ,并利用 pET16b表达载体在大肠杆菌BL2 1(DE3)菌株中高效表达wbbL基因产物 -鼠李糖基转移酶。 [方法 ]利用PCR方法从结核分枝杆菌H 37Rv菌株的基因组DNA中扩增出wbbL基因 (90 6bp)并克隆到 pMD18-T克隆载体中 ,经DNA序列测定证实为正确的基因后 ,将其亚克隆到表达载体 pET16b中并在大肠杆菌BL2 1(DE3)中表达wbbL基因产物。 [结果 ]1)从结核分枝杆菌H37Rv菌株的基因组DNA中PCR扩增出正确的wbbL基因 ;2 )经IPTG诱导wbbL基因在BL2 1(DE3)中高效表达出wbbL基因产物。 [结论 ]1)用具高保真性的LATaqDNA聚合酶所扩增的结核分枝杆菌的wbbL基因经BLAST分析与结核分枝杆菌基因组数据库 (http :/ /www .sanger .ac .uk)中所公布的序列完全一致 ;2 )从大肠杆菌BL2 1(DE3)所获得的大量鼠李糖基转移酶 ,为进一步纯化该酶并研究其酶促反应动力学特性提供了物质保障。

Abstract:

[Objective] To clone wbbL gene from M.tuberculosis H37Rv and to overexpress WbbL protein in E.coli BL21(DE3) by using pET16b expression vector. [Methords] The gene encoding rhamnosyl transferase was amplified by polymerase chain reaction (PCR) from genomic DNA of M.tuberculosis H37RV strain and cloned into a cloning vector pMD18-T for resequencing wbbL gene. The wbbL gene was subcloned to an expression vector pET16b and recombinant wbbL was overexpressed in E.coli BL21(DE3). [Results] 1)The wbbL gene was amplified successfully from M.tuberculosis H37Rv genomic DNA by PCR. 2) The WbbL protein was overexpressed in E.coli BL21(DE3) after 1mM IPTG induction at 37 ℃. [Conclusion] The DNA sequence of wbbL was identical with the H37Rv genome database in Sanger (http://www.sanger.ac.uk).