结核分枝杆菌Ag85B基因体外扩增、克隆及在耻垢分枝杆菌中的表达

目的体外扩增结核杆菌Ag85B基因,构建大肠埃希菌-分枝杆菌穿梭质粒ps3000-Ag85B,并重组耻垢分枝杆菌(Mycobacteriums megmatis mc2155)。方法采用聚合酶链式反应(PCR)方法,体外扩增结核杆菌Ag85B基因,克隆入pGEMT载体;构建亚克隆ps3000-Ag85B大肠埃希菌-分枝杆菌穿梭质粒,电转化方法将有Ag85B基因的穿梭质粒转化到耻垢分枝杆菌中。热诱导此重组耻垢分枝杆菌,用SDS-PAGE电泳观察Ag85B蛋白的表达,Western blot鉴定其生物学活性。结果PCR扩增结核杆菌Ag85B基因片段大小为990bp,构建的穿梭质粒ps3000-Ag85B酶切片段为990bp,与理论值相符。经Western blot检测,该重组耻垢杆菌表达蛋白能被结核病患者血清识别。结论Ag85B重组耻垢分枝杆菌构建成功,为下一步表达Ag85B蛋白的重组BCG(Bacilli Calmette-Guérin)疫苗的研究奠定了基础。

Amplification, cloning of Ag85B gene from Mycobacterium tuberculosis and its expression in Mycobacterium smegmatis mc2155

Objective To amplify Ag85B gene of Mycobacterium tuberculosis, and construct Escherichia coli-Mycobacterium shuttle plasmid ps3000-Ag85B and recombinant M. smegmatis mc2155. Methods The Ag85B gene was amplified from the genomic DNA by polymerase chain reaction. And the cloning vector PGEMT-Ag85B and shuttle vector ps3000-Ag85B were constructed, which was transformed into M. smegmatis mc2155 by electroporation. The recombinant M. smegmatis mc2155 was induced by heating and the expression and activity of the target protein were determined by SDS-PAGE and immunoblotting. Results The fragment of Ag85B gene amplified from the genomic DNA by polymerase chain reaction was 990 bp, and the shuttle vector ps3000-Ag85B was constructed, which was demonstrated by digestion of restrictive enzyme.The target protein could be expressed and recognized by antiserum of TB patients. Conclusion The recombinant M. smegmatis mc2155 is successfully constructed,which may pave the way for further study of recombinant BCG vaccine expressing Ag85B protein.