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大肠杆菌胸苷激酶基因的克隆表达与应用研究

Cloning,Expression and Application of Thymidine Kinase from Escherichia coli K-12
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摘要 目的:构建含大肠杆菌胸苷激酶基因(tdk)的重组质粒,并对胸苷激酶性质进行研究。方法:通过PCR技术从大肠杆菌K-12中扩增出tdk基因,并克隆到表达载体pET-28a(+)上,转入大肠杆菌BL21(DE3)中构建重组菌株(DTK)。结果:成功扩增出了tdk基因,并构建了重组菌株。结论:获得的tdk基因长为618bp,重组菌株能表达出大量可溶性胸苷激酶蛋白。胸苷激酶的酶比活力可达到1 600U/mg;酶最适温度为45℃;最适pH为8.0。同时,胸苷激酶最佳反应条件为:50mmol/L pH 8.0的Tris-HCl缓冲液,30U的粗酶,5mmol/L的MgCl2,5mmol/L的TR,5mol/L的ATP,45℃反应100min,胸苷酸的转化率可达到60%以上。 Objective:Construct the recombinant plasmid containing the gene encoding thymidine kinase from Escherichia coli K - 12 and study the properties of thymidine kinase. Method:The sequence of tdk gene was amplified by PCR from Escherichia coli K - 12 and was cloned into expression vector pET-28a( + ), then the recombinant plasmid was transformed into the strain E. coli BL1 ( DE3 ). Result: The tdk gene was amplified and the recombinant strain was constructed successfully. Conclusion:The tdk gene was 618 bp in length and thymidine kinase was highly expressed. The specific activity of thymidine kinase reached to 1 600U/mg. The optimum temprature was 45℃ and pH was 8. 0. The optimum condition of reaction catalyzed by thymidine kinase was 50mmolZL Tris - HCI (pH 8. 0), 30U enzyme, 5mmolZL MgC12 ,Smmol/L TR,SmolZL ATP. The conversion rate of thymidylate could reach to 60% after 100 min at 45℃.
出处 《生物技术》 CAS CSCD 北大核心 2013年第5期20-24,共5页 Biotechnology
关键词 胸苷 胸苷酸 胸苷激酶 Thymidine Thymidylate Thymidine kinase
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