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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 a biologically active Mycobacterium tuberculosis recombinant isocitrate lyase protein. Methods Mycobacterium tuberculosis H37Rv genome was used as a template to amplify the isocitrate lyase gene ic 1 and cloned into prokaryotic expression vector pET-28 a (+). The recombinant plasmid was cloned into E. coli BL21 (DE3) The recombinant protein purified by nickel ion chelating agarose gel affinity chromatography was obtained and its enzymatic properties were determined. Results The recombinant Mycobacterium tuberculosis isocitrate lyase with biological activity was purified. The analysis of enzymatic properties showed that the specific activity of the recombinant isocitrate lytic enzyme ICL was 7.657 × 102μmol.mg-1.m in-1, and the optimal reaction pH was about 7.4. The recombinant protein was identified by high performance liquid chromatography and mass spectrometry. The relative molecular mass was 50 603.347. In the 5 mmol / L Tris-Cl buffer at pH 7.8,25 ℃, the relative secondary structure of recombinant ICL was 43.8% α-helix, 31.9% β-sheet, 3.4% β-turn, 20.9% Curly rules. Conclusion In this study, we cloned and expressed the isocitrate lyase gene of Mycobacterium tuberculosis H37Rv successfully and obtained the biologically active recombinant protein. This study laid the foundation for the study of immunoenzyme and the screening of novel antituberculosis drugs.