结核分枝杆菌异柠檬酸裂解酶基因的克隆表达及酶学性质研究

Molecular cloning,expression,purification and properties of isocitrate lyase gene from Mycobacterium tuberculosis H_(37)Rv

目的获得具有生物学活性的结核分枝杆菌重组异柠檬酸裂解酶蛋白。方法以结核分枝杆菌H37Rv基因组为模板,扩增该菌株的异柠檬酸裂解酶基因ic l,克隆入原核表达载体pET-28 a(+)中,通过在大肠杆菌BL21(DE3)中表达,获得以镍离子螯合型琼脂糖凝胶亲和层析柱纯化的重组蛋白,并对其酶学性质进行测定分析。结果纯化出具有生物学活性的重组结核分枝杆菌异柠檬酸裂解酶。酶学性质测定分析表明,重组异柠檬酸裂解酶ICL的比活力为7.657×102μmol.mg-1.m in-1,反应最适pH值约为7.4。重组蛋白经高效液相色谱及质谱鉴定,测得相对分子质量为50 603.347。在5 mmol/L Tris-C l缓冲液、pH值7.8、25℃条件下,重组ICL的二级结构中相对有43.8%的α螺旋、31.9%的β折叠、3.4%β转角、20.9%无规则卷曲。结论本研究成功克隆表达结核分枝杆菌H37Rv异柠檬酸裂解酶基因,酶学性质鉴定获得了具有生物学活性的重组蛋白,为该酶免疫学研究及新型抗结核药物的筛选奠定了基础。

Objective To obtain recombinant protein with enzymatic activities of isocitrate lyase （ICL）. Methods The icl gene was amplified by polymerase chain reaction （PCR） from Mycobacterium tuberculosis H37Rv strain genomic DNA and cloned into pET28-a（ ＋ ） vector. The recombinant protein was expressed in E. coli BL21 （ DE3 ）. Enzyme activity of the protein was assayed after purifing with Ni-NTA resin. Results The recombinant ICL was purified in a highly active state with a specific activity of about 7. 657 × 10^2 μmol · mg^-1·min^-1. The pH curve indicated that recombinant ICL activity was optimal at pH 7.4. The LC/MS spectrometry showed a 50 603. 347 molecular mass of recombinant ICK The CD spectrum showed that the percentages for α- helix, β- sheet, β- turn ,and random coil were 43. 8 % ,31.9% ,3. 4% , and 20.9 %, respectively. Conclusions The icl gene of Mycobacterium tuberculosis H37 Rv was successfully cloned and expressed. The enzymatic properties demonstrated the purified recombinant protein had activities of ICK This work can facilitate immunologic research and the discovery of novel antimicrobial agents against Mycobacterium tuberculosis.