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目的构建含有His标签结核分枝杆菌esat-6基因的原核表达质粒,并在体外进行原核表达和纯化。方法提取结核分枝杆菌基因组DNA,对其采用聚合酶链反应技术扩增esat-6基因,并构建至表达载体p ET28a上。将测序正确的载体转化大肠杆菌BL21,然后对His-ESAT6融合蛋白通过IPTG诱导进行表达,最后对重组蛋白使用聚丙烯酰胺凝胶电泳和免疫印迹进行分析。结果 PCR扩增出结核分枝杆菌esat-6基因,成功构建至表达载体p ET28a-ESAT-6上,并在体外优化表达,产生高水平的目的蛋白表达产物。结论成功构建并表达了含有His标签的ESAT-6融合蛋白,为临床中预防和治疗结核病提供了有效的参考。
Objective To construct a prokaryotic expression plasmid containing the His tag-tagged Mycobacterium tuberculosis esat-6 gene and prokaryotic expression and purification in vitro. Methods Mycobacterium tuberculosis genomic DNA was extracted, and esat-6 gene was amplified by polymerase chain reaction (PCR) and cloned into expression vector p ET28a. The correctly sequenced vector was transformed into E. coli BL21, and then the His-ESAT6 fusion protein was induced by IPTG. Finally, the recombinant protein was analyzed by polyacrylamide gel electrophoresis and Western blotting. Results The Mycobacterium tuberculosis esat-6 gene was amplified by PCR and successfully cloned into expression vector pET28a-ESAT-6. The recombinant plasmid was optimized for expression in vitro and produced a high level of expression product of the target protein. Conclusion The His tag-containing ESAT-6 fusion protein was successfully constructed and expressed, which provided an effective reference for the prevention and treatment of tuberculosis in clinic.