用PCR方法定向突变结核分枝杆菌glmU基因

Site-specific Mutation of M. tuberculosis by PCR

目的:结核分枝杆菌glmU基因是分枝杆菌生长必需基因,其编码产物具有乙酰基转移酶活性和尿嘧啶转移酶活性,参与细胞壁前体物UDP-乙酰葡糖胺(UDP-GlcNAc)的生物合成,研究其空间结构可以定向设计酶抑制剂。方法:利用PCR方法定向突变结核分枝杆菌glmU基因,并用大肠杆菌BL21(DE3)高表达m-GlmU蛋白。结果:获得了定向突变的结核分枝杆菌glmU基因,m-glmU。纯化的m-GlmU蛋白仍具有乙酰基转移酶活性和尿嘧啶核苷转移酶活性。结论:纯化的m-GlmU蛋白为进一步研究其稳定性、测定其空间结构提供了物质基础。

Objective： M. tuberculosis glmU gene is an essential gene for mycobacterial growth. The GlmU protein encoded by the glmU gene is a bi-functional enzyme which exhibits uridyltransferase and acetyltransferase and involves in the biosynthesis of UDP-GlcNAc, a precursor of cell wall. It is necessary to study its crystal structure and design its inhibitors. Method： The M. tuberculosis glmU was cloned and the site specific mutagenesis of glmU gene was generated by PCR. The m-GlmU protein was over-expressed in E. coli BL21 （DE3）. Result： The mutant glmU gene, m-glmU, was confirmed and the uridyltransferase and acetyltransferase activities of the purified m-GlmU protein were detected. Conclusions： The purified m-GlmU protein will be utilized for detection of its stability and analysis of its three-dimensional structure.