结核杆菌H37Rv株重组KatG蛋白的表达纯化研究  

Study on expression and purification of Mycobacteriuml tuberculosis recombinant KatG

作　　者：石君帆[1] 宋广忠[1] 漏磊君[1] 杨明瑾[1] 沈丽英[1] 曾肖芃[1] John T．Belisle 

机构地区：[1]浙江省医学科学院,浙江杭州310013 [2]美国科罗托多大学

出　　处：《中国病原生物学杂志》2008年第1期12-15,共4页Journal of Pathogen Biology

基　　金：浙江省自然科学研究基金资助项目(No.Y204480)

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。Objective To express and purify isoniazid（INH） resistance associated catalase-peroxidase KatG gene from Mycobacterium tuberculosis in Escherichia coll. 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.

关 键 词：KATG 耐药机制 亚细胞定位 结核分枝杆菌 

分 类 号：R378.91[医药卫生—病原生物学]