结核分枝杆菌PPE19的克隆及其GST融合蛋白的表达和鉴定

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。

Expression and identification of Mycobacterium tuberculosis PPE19-GST fusion protein

Objective To build the expression vector of Mycobacterium tuberculosis PPE19-GST fusion protein and identify its expression in E.coli.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 that the recombinant plasmids were correct.The recombinant GST-PPE19 expression was about 70 kDa,in the form of solution in E.coli DH5α.Conclusion The recombinant PPE19 proteins were successfully expressed,which laid foundation for further function study of PPE19 of Mycobacterium tuberculosis.