飞蝗羧酸酯酶基因克隆及真核表达 - 应用昆虫学报.docVIP

飞蝗羧酸酯酶基因Lm，张学尧1，张建琴1，张建珍1，郭亚平2 马恩波1** （1山西大学应用生物学研究所, 2 山西大学生命科学学院，太原 030006） 摘要 羧酸酯酶（Carboxylesterase, CarE）是一类广泛分布的多功能家族酶系，在昆虫抗药性形成机制中发挥重要作用。为探讨飞蝗Locusta migratoria羧酸酯酶LmCarE25的功能，在飞蝗成虫各组织部位均有表达，胃盲囊中表达量较低，、马氏管和脂肪体表达量较高。尝试在大肠杆菌和昆虫细胞体外重组表达该构建重组表达载体pET32-LmCarE25，LmCarE25可在大肠杆菌BL21(DE3)和JM109中表达，蛋白分子量为60 kDa，但为包涵体；将LmCarE25基因插入真核表达载体pFastBacHTA中，Bac-to-Bac?体系获得重组sf9细胞系，Western-Blot检测结果显示，LmCarE25可溶性表达。上述结果为进一步深入研究飞蝗羧酸酯酶家族功能。 关键词：飞蝗，羧酸酯酶，eal-time PCR，原核表达，真核表达 xpression of carboxylesterase gene LmCarE25 from Locusta migratoria WANG Jian-Xin1, ZHANG Xue-Yao1, ZHANG Jian-Qin1, ZHANG Jian-Zhen1, Guo Ya-ping2 MA En-Bo1* (1 Institute of Applied Biology, Shanxi University, 2 College of Life Science, Shanxi University, Taiyuan 030006, China) Abstract：Carboxylesterases (CarE) insecticide resistance. In this present study the physiological functions of the carboxylesterase member LmCarE25 was investigated from Locusta migratoria(Meyen). Real-time PCR rresults showed that the LmCarE25 was expressed in all of the adult tissues, while had lower expression levels in gastric cecum, wings and muscles, and higher expression levels in midgut, Malpighian tubules and fat body. In addition, colibacillus and insect cell expression system were used to express this enzyme in vitro. The recombinant LmCarE25 was successfully expressed by recombinant vector pET32a-LmCarE25 in E. coli BL21(DE3) and JM109, and its molecular weight was approximately 60 kDa, although it was inclusion bodies. Then LmCarE25 was subcloned into eukaryotic expression vector pFastBacHTA, the recombinant bacmid DNA was obtained by Bac-to-Bac? system, and was transfected into sf9 cells by lipid mediated transfection. Western-blot results suggested LmCarE25 expressed in soluble form at low level. These results might provide information for further study the function of carboxylesterase family of Locusta migratoria. Keywords: Locusta migratoria, Carboxylesterase, Real-time PCR, Prokaryotic expression, Eukaryotic expressi