结核分枝杆菌PPE19的克隆及其GST融合蛋白的表达和鉴定  

Expression and identification of Mycobacterium tuberculosis PPE19-GST fusion protein

作　　者：熊志红[1] 朱琰[1] 李仁德[1] 庄玉辉[1] 程小星[1] 

机构地区：[1]解放军第309医院结核病研究室,北京100091

出　　处：《临床肺科杂志》2012年第1期77-79,共3页Journal of Clinical Pulmonary Medicine

基　　金：国家重大传染病专项基金资助项目(基金编号:2008ZX10003-012)

摘　　要：目的构建结核分枝杆菌PPE19的GST融合蛋白表达载体,并鉴定其在大肠杆菌中的表达。方法以结核杆菌H37Rv基因组为模板,根据PPE19基因序列设计引物,运用PCR的方法获得PPE19基因,并将其克隆到pGEX-kg载体中,转化大肠杆菌DH5α,挑取阳性菌落进行质粒的酶切和序列分析;将构建正确的克隆进行诱导表达,对菌体裂解物进行SDS-PAGE分析;用western-blot鉴定融合蛋白的表达。结果正确构建了pGEX-PPE19原核表达载体,载体上基因序列正确;载体能在大肠杆菌中表达分子量约为70 kDa的重组蛋白;该蛋白能与GST抗体结合。结论成功构建了GST-PPE19融合蛋白的表达载体,为进一步进行PPE19蛋白的功能研究奠定了基础。Objective To build the expression vector of Mycobaeterium tuberculosis PPE19-GST fusion protein and identify its expression in E, colt. Methods The PPE19 gene was amplified from H37Rv genome using PCR method, then cloned into pGEX-kg vector. The expressive vector pGEX-PPE19 was transformed into E. coli DH5α. The expression of GST-PPE19 fusion protein was detected by SDS-PAGE and Werstern-blot. Results The result of restriction enzyme digestion and nucleotide sequencing confirmed thai the recombinant plasmids were correct. The recombinant GST-PPE19 expression was about 70 kDa, in the fnrm of solution in E. colt DH5α. Conclusion The recombinant PPE19 proteins were successfully expressed, which laid foundation for further function study of PPEI9 of Mycobacterium tuberculosis.

关 键 词：结核分枝杆菌 PPE19 GST融合蛋白 表达与鉴定 

分 类 号：R52[医药卫生—内科学]