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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 recombinant KatG (rKatG) protein of Mycobacterium tuberculosis H37Rv and lay a foundation for further study on the mechanism of isoniazid resistance. Methods The recombinant plasmid pET23b-KatG was transformed into Escherichia coli BL21 (DE3) plysE and IPTG induced the expression of rkatG. After SDS-PAGE electrophoresis and Western blot, the expression of rKatG protein was optimized by nickel ion chelate affinity chromatography, and the catalase activity of the purified product was determined. Results The recombinant plasmid pET23b-KatG was successfully constructed. The rKatG protein was expressed as a soluble protein. The purity of rKatG protein was 95.6% after affinity chromatography, and the catalase test was positive. Conclusion The constructed pET23b-KatG recombinant plasmid can efficiently express the soluble rKatG protein with enzyme activity, and the purified protein can be obtained by affinity chromatography.