结核杆菌H37Rv异柠檬酸裂解酶基因的克隆及表达

目的构建在大肠杆菌中高效表达结核杆菌H37Rv异柠檬酸裂解酶(ICL)的重组质粒,实现ICL在原核表达系统的高效稳定表达.方法采用PCR和克隆技术构建含有结核杆菌H37Rv ICL基因的表达质粒pET30(a)-Rv0467,转化至E.coli BL21(DE3)中诱导表达.目的蛋白经金属螯合层析纯化后,对其活性进行初步研究.结果构建了高效表达结核杆菌H37Rv ICL的质粒;在E.coli BL21(DE3)中得到高效表达,目的蛋白占总蛋白含量的30%;表达产物以可溶性形式存在,通过金属螯合层析纯化,所得酶的纯度为90%;目的蛋白具有ICL的活性.结论利用原核表达系统成功克隆表达了结核杆菌H37Rv的ICL,为以ICL为靶点建立新型抗结核药物奠定了基础.

Clone and expression of isocitrate lyase gene in Mycobacterium tuberculosis H37Rv

Objective To construct recombinant plasmid with isocitrate lyase (ICL) gene of Mycobacterium tuberculosis H37Rv for stable and high level expression of ICL in prokaryotic expression system. Methods The recombinant plasmid with ICL gene (pET30 (a)-Rv0467) was constructed by polymerase chain reaction and cloning. The fusion protein was expressed in E.coli host strain BL21 (DE3). Activity of the fusion protein was studied after it was purified with metal chelating chromatography. Results We constructed the plasmid which could highly express Mycobacterium tuberculosis H37Rv ICL. The plasmid was highly expressed in E.coli BL21 (DE3), in which the fusion protein accounted for 30% of total protein content. After having been purified by metal chelating chromatography, the purity of the soluble fusion protein was 90%. The fusion protein had activity of ICL. Conclusion Using the prokaryotic expression system, the ICL gene of Mycobacterium tuberculosis H37Rv was successfully cloned and expressed, which build the basis for screening new anti-tuberculosis drugs with ICL as the target point.