结核分枝杆菌ESAT-6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ESAT-6基因,并构建大肠杆菌-分枝杆菌穿梭质粒ps3000-ESAT-6和重组耻垢分枝杆菌。方法采用聚合酶链式反应(PCR)方法,体外扩增结核杆菌ESAT-6基因,克隆入pGEMT载体。然后,构建亚克隆ps3000-ESAT-6大肠杆菌-分枝杆菌穿梭质粒,经电转化方法,将ESAT-6基因的穿梭质粒转化到耻垢分枝杆菌(Mycobacteri-umsmegmatis mc~2155)中,热诱导此重组耻垢分枝杆菌,用SDS-PAGE电泳观察ESAT-6蛋白的表达,Western blotting鉴定其生物学活性。结果成功扩增了结核杆菌ESAT-6基因;正确构建穿梭质粒ps3000-ESAT-6,用pET表达系统ESAT-6纯化蛋白免疫小鼠的多抗血清通过Western blot证实了该重组耻垢杆菌中有表达并具有生物学活性。结论ESAT-6重组耻垢分枝杆菌构建成功,为下一步表达ESAT-6蛋白的重组BCG(BacilliCalmette-Guérin)疫苗的研究奠定了基础。

In vitro amplification,cloning of Mycobaterium tuberculosis ESAT-6 gene and its expresson in Mycobacterium smegmatis mc2 155

To amplify ESAT-6 gene of Mycobaterium tuberculosis and construct E.coli-Mycobacterium shuttle plasmid ps3000-ESAT-6 and recombinant M.smegmatis mc2 155. The ESAT-6 gene was amplified from the genomic DNA by polymerase chain reaction in vitro. And the cloning vector pGEMT-ESAT-6 and shuttle vector ps3000-ESAT-6 which was transformed into M.smegmatis mc2 155 by electroporation. The recombinant M.smegmatis mc2 155 was induced by heating, and then the expression and activity of the target protein were determined by SDS-PAGE and immunoblotting. The ESAT-6 gene was amplified from the genomic DNA by polymerase chain reaction. And shuttle vector ps3000-ESAT-6 was correctly construced. The target protein could be expressed and recognized by the mice antiserum against the purified protein pET-ESAT-6. It was demonstrated that the recombinant M.smegmatis mc2 155 could expressed the target protein with biologic activity.In conclusion, the recombinant M.smegmatis mc2 155 was successfully constructed and pave the way for further study of recombinant BCG vaccine expressing ESAT-6 protein.