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资料包括： 论文(4页3092字)
说明：
【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 mmol/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。[ HTH〗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。
关键词： 分支杆菌,结核,katG,基因表达,蛋白纯化
Overexpression and purification of catalase-peroxidase katG from mycobacterium tuberculosis
【Abstract】Objective To express and purify the catala se-peroxidase katG gene from mycobacterium tuberculosis. Methods Plasmid pET24b containing katG was transferred into com petent Escherichia coli and katG gene was overexpressed by the challenge of isopropylthio-β-D-glactoside(IPTG). The expression of katG protein was on e-step purified by Xpress systemTM. Furthermore, the catalase activity of katG protein was preliminarily detec ted. Results The recombinant escherichia coli produced katG p rotein in large quantities, accounting for 17.7% of total cell protein. The mole cular 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. Th e katG protein was preliminarily detected to have the activity of catalase. Conclusions The stable katG overexpressing recombinant Escherichi a coli can be constructed by the plasmid pET24b containing katG gene. The reco mbinant strain can produce katG protein with catalase activity and the product o f which can be purified into higher activity.
【Key words】Mycobacterium tuberculosis; katG ; Gene expression; Protein purification
目录：
一、材料
二、方法
结 果
参考文献：
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