【摘要】 目的表达纯化结核杆菌H37Rv株重组KatG(简称rKatG)蛋白,为深入研究异烟肼耐药机制奠定基础。方法重组质粒pET23b-KatG转化大肠埃希菌表达菌株BL21(DE3)plysE,IPTG诱导rkatG蛋白表达。经SDS-PAGE电泳和Western blot鉴定后,优化表达条件,用镍离子鳌合亲和层析柱纯化rKatG蛋白,对纯化产物进行过氧化氢酶活性测定。结果成功构建了重组质粒pET23b-KatG,rKatG蛋白以可溶性蛋白形式表达,经亲和层析后的rKatG蛋白纯度为95.6%,过氧化氢酶试验阳性。结论构建的pET23b-KatG重组质粒能高效表达有酶活性的可溶性rKatG蛋白,经亲和层析后可得到高纯度的纯化蛋白。更多还原

【Abstract】 Objective To express and purify isoniazid(INH) resistance associated catalase-peroxidase KatG gene from Mycobacterium tuberculosis in Escherichia coli.Methods Recombinant plasmid pET23b-KatG was transformed into E.coli BL21(DE3)plysE,the recombinant KatG(rKatG) Protein was expressed in E.coli BL21(DE3) plysE by induction with IPTG.After identification by SDS-PAGE electrophoresis and western blot,the expression condition of rKatG was optimized.Then rKatG was purified by nickel-chelate affinity chromatography.Furthermore,the catalase activity of rKatG was preliminarily detected.Results A recombinant plasmid pET23b-KatG has been successfully constructed and can stably express a 80 ku rKatG,existed in soluble form.The purity of rKatG reached 95.6% after affinity chromatography.The rKatG was preliminarily detected to have the activity of catalase.Conclusion The successfully constructed pET23b-KatG can highly express soluble rKatG Protein with catalase activity,the high-purity rKatG can be obtained by affinity chromatography.更多还原