重组葡萄糖脱氢酶基因的表达研究  

Study on the Expression of Recombinated Glucose Dehydrogenase Gene

作　　者：周丽萍[1] 姜旭淦[1] 徐顺高[1] 郑铁生[1] 王卉放[1] 

机构地区：[1]江苏大学医学技术学院生物化学教研室,江苏镇江212001

出　　处：《江苏大学学报（医学版）》2005年第4期291-293,共3页Journal of Jiangsu University：Medicine Edition

基　　金：江苏大学青年基金资助项目(02JDQ030)

摘　　要：目的:将重组的葡萄糖脱氢酶基因在大肠杆菌中表达,优化表达条件,以期获得较高活力的葡萄糖脱氢酶.方法:将枯草芽孢杆菌葡萄糖脱氢酶基因与表达载体pET22b连接后转化至大肠杆菌JM109(DE3)进行表达,经对发酵时间、诱导物浓度、诱导时间以及细胞破碎等条件的控制,实现葡萄糖脱氢酶的高表达.结果:重组质粒转化菌发酵2 h后进入对数生长期,诱导剂IPTG浓度为0.5 mmol/L,诱导2.5～3 h后用240 W超声破碎细胞,表达的粗提酶活力比诱导前提高了近千倍.SDS-PAGE电泳显示重组菌能表达单体分子量为31.5KD的特异性蛋白.结论:表达质粒的拷贝数、宿主菌培养条件、细胞破碎方式等均能影响酶的表达量.Objective： To express the recombinated glucose dehydrogenase gene in E. coli, and to optimize expression conditions in order to gain the high activity enzyme. Methods： The gene for the GDH from B. subtilis was ligated with vector pET22b, then transducted into E. coli JM109 and expressed. The expression of high activity for GDH was studied through the control of conditions such as culture time, inducer concentration, inductiontime and cell disrepution. Results： The recombinated plasmid entered up to logphase after 2 hours culture. The optimal IPTG concentration was 0.5mmol/L and optimal induction time was 2.5 - 3.0 h. The activity of the crude GDH after adopting 240w cell sonication was nearly 1000 times higher compared with no inducer. The subunit molecular weight of GDH was 31. 5kDal by SDS-PAGE. Conclusion ： The culture conditions and procedure of cell disrepution can influence the expression of GDH.

关 键 词：葡萄糖脱氢酶 表达 基因 

分 类 号：Q75[生物学—分子生物学] R394.2[医药卫生—医学遗传学]