结核分枝杆菌突变gyrase A基因原核表达载体的构建与鉴定

目的构建结核分枝杆菌Ser95Thr突变DNA促旋酶A(DNA gyrase A)基因原核表达载体并鉴定,为进一步研究奠定基础。方法以结核分枝杆菌H37Rv基因组为模板,应用重叠延伸剪切技术,分别通过3次PCR扩增Ser95Thr突变gyrase A基因编码序列,定向克隆入融合蛋白原核表达载体pET-32 a(+),获得重组表达质粒pET-mgyr。结果从结核分枝杆菌H37Rv株基因组DNA中扩增出Ser95Thr突变gyrase A基因,经过酶切、PCR和测序鉴定,表明突变gyrase A基因正确地插入原核表达载体pET-32 a(+)。结论成功构建了原核表达载体pET-mgyr,为Ser95Thr突变gyrase A基因的功能研究奠定了基础。

Construction and Identification of Prokaryotic Expression Vector Bearing the Mutant Gyrase A gene From Mycobacterium Tuberculosis

Objective To construct and identify a Ser95Thr mutant gyrase A gene from Mycobacterium tuberculosis and provide materials for investigating the function of the gene.Methods The Ser95Thr mutant gyrase A gene of Mycobacterium tuberculosis was amplified by the splicing by overlapping extension(SOE)-PCR and cloned into prokaryotic expression vector pET-32 a(+).The recombinant plasmid pET-mgyr was transformed into E.coli DH5α and identified by restriction endonuclease,PCR and sequencing.Results The Ser95Thr mutant gyrase A gene was amplified accurately from the genome DNA of H37Rv.The mutant gene was correctly cloned into prokaryotic expression vector pET-32a(+).Conclusion The prokaryotic expression vector pET-mgyr was successfully constructed.It provided the basis for the further study of the Ser95Thr mutant gyrase A gene.