结核分枝杆菌H_(37)Rv异柠檬酸裂解酶基因的克隆表达及活性

Cloning,Expression and Properties of Isocitrate Iyase Gene in Mycobacterium tuberculosis H_(37)Rv

以结核分枝杆菌H37Rv基因为模板,PCR反应扩增该菌株的异柠檬酸裂解酶基因(ICL),将其克隆入原核表达载体pET28b中,并将pET28b-ICL转化入大肠杆菌BL21(DE3)中进行诱导表达.结果表明,ICL蛋白的最佳诱导表达条件为:温度20℃,IPTG终浓度为0.25 mmol/L,诱导表达4 h,在此条件下ICL实现了高效表达,以镍离子螯合型琼脂糖凝胶亲和层析柱纯化ICL蛋白,纯化程度较高.酶学性质鉴定表明,实验获得了具有生物学活性的重组蛋白,重组ICL的比活力为24μmol/(mg.min).

Isocitrate lyase（ICL） gene was amplified by polymerase chain reaction（PCR） from template Mycobacterium tuberculosis H37Rv strain genomic DNA and was cloned into expression vector pET28b,named pET28b-ICL.The recombined plasmid pET28b-ICL was transformed into E.coli BL21（DE3）.The optimum conditions for ICL expression in E.coli BL21（DE3） were determined to be 0.25 mmol/L IPTG induction for 4 h at 20 ℃.The expressed ICL was further purified by means of Ni-NTA resin affinity chromatography.The recombinant ICL was purified in a highly active state with a specific activity of 24 μmol/（mg·min）.