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。 Objective: To clone and express Mycobacterium tuberculosis ESAT-6. Methods: The genomic DNA of Mycobacterium tuberculosis H37Rv was used as template to amplify the esat-6 gene fragment by polymerase chain reaction (PCR) and cloned into pMD18-T vector. The positive clones were screened by PCR and sequenced. The esat-6 gene was subcloned into pET-28a to construct the recombinant plasmid pET-esat-6. The recombinant plasmid was transformed into E. coli BL21 and identified by PCR and restriction enzyme digestion. The recombinant plasmid was induced by IPTG, Column purification ESAT-6, SDS-PAGE and Western blotting identification. Results: The specific fragments of esat-6 gene were amplified by PCR. The recombinant plasmid pET-esat-6 was successfully constructed and expressed in BL21. The purified ESAT-6 protein was identified by the serum of tuberculosis patients. Conclusion: The recombinant protein ESAT-6 was successfully cloned and expressed.