【摘要】 目的:克隆和表达结核分枝杆菌ESAT-6。方法:以结核分枝杆菌H37Rv基因组DNA为模板,利用聚合酶链式反应(PCR)扩增esat-6基因片段,克隆至pMD18-T载体,PCR筛选阳性克隆并测序。将esat-6基因亚克隆至pET-28a,构建pET-esat-6重组质粒,转化入大肠杆菌BL21感受态,PCR和双酶切鉴定阳性克隆,经IPTG诱导表达,用His-bindTM亲和层析柱纯化ESAT-6,SDS-PAGE和Western blotting鉴定。结果:PCR扩增出esat-6基因的特异片段,成功构建重组表达质粒pET-esat-6,并在BL21获得表达,纯化的ESAT-6能被结核病人血清所识别。结论:成功克隆和表达获得重组蛋白ESAT-6。更多还原

【Abstract】 Objective To clone and express Mycobacterium tuberculosis ESAT-6 recombinant protein.Methods esat-6 gene was amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA and cloned into the pMD18-T vector. The positive clones were identified by PCR and sequencing. esat-6 was inserted into pET-28a vector. and transformed into E.coli BL21. The positive clones were identified by PCR and double digestion. Recombinant protein was expressed in the positive clones when induced with IPTG, the expressed ESAT-6 recombinant protein was purified by His-bindTM affinity chromatography and confirmed by SDS-PAGE and Western blotting. Results Esat-6 gene fragment was amplified from genomic DNA of Mycobacterium tuberculosis H37Rv strain, the pET-esat-6 prokaryotic recombinant plasmid was constructed. The recombinant protein expressed in E.coli BL21 was purified and could be recognized by sera of patients infected with Mycobacterium tuberculosis. Conclusion The recombinant plasmid expressed ESAT-6 protein was successfully constructed.更多还原