【摘要】 目的　表达与纯化结核分支杆菌katG 蛋白,为深入研究异烟肼耐药机制奠定基础。方法　将含有katG 基因的pET24bkatG 表达载体转化大肠杆菌BL21(DE3) 菌株,在异丙基硫代βD半乳糖苷(IPTG) 诱导下表达,分别对不同诱导时间的表达产物进行SDSPAGE 以及考马斯亮蓝染色。获得稳定的高表达菌株后采用Xpress TM蛋白纯化系统对超声破菌液进行纯化。最后对纯化产物进行过氧化氢酶活性的初步检测。结果　对诱导后的重组大肠杆菌菌液进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDSPAGE) 以及考马斯亮蓝染色后发现相对分子质量约为80 000 。表达蛋白量约占总蛋白量的17.7% 。对重组katG基因表达产物进行纯化的结果发现,以350 m mol/L咪唑洗脱时的纯化效果最理想,蛋白纯度可达90 % 以上。对表达产物进行过氧化氢酶活性初步检测证明,重组的katG 基因产物具有过氧化氢酶活性。结论　通过pET24bkatG 表达质粒转化大肠杆菌可获得基因重组的katG 高表达菌株,表达产物具有一定酶活性,经纯化后可达到较高的纯度更多还原

【Abstract】 Objective To express and purify the catalase peroxidase katG gene from mycobacterium tuberculosis. Methods Plasmid pET24b containing katG was transferred into competent Escherichia coli and katG gene was overexpressed by the challenge of isopropylthio β D glactoside(IPTG). The expression of katG protein was one step purified by Xpress system TM . Furthermore, the catalase activity of katG protein was preliminarily detected. Results The recombinant escherichia coli produced katG protein in large quantities, accounting for 17.7% of total cell protein. The molecular mass of katG protein was estimated to be 80 000 by sodium dodecyl sulfate polyacrylamide gradient gel electrophresis (SDS PAGE). It was found that imidazole at the concentration of 350 mmol/L could elute the katG protein most efficiently and yield the final preparation at greater than 90% purity. The katG protein was preliminarily detected to have the activity of catalase. Conclusions The stable katG overexpressing recombinant Escherichia coli can be constructed by the plasmid pET24b containing katG gene. The recombinant strain can produce katG protein with catalase activity and the product of which can be purified into higher activity.更多还原