结核分枝杆菌H37Rv异柠檬酸裂解酶基因的克隆表达及活性

以结核分枝杆菌H₃₇Rv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 I
CL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24 μmol/(mg·min).

Cloning, Expression and Properties of Isocitrate Iyase Gene in Mycobacterium tuberculosis H37Rv

Isocitrate lyase(ICL) gene was amplified by polymerase chain reaction (PCR) from template Mycobacterium tuberculosis H₃₇R
v 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 wasfurther 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).