大肠杆菌-枯草芽孢杆菌穿梭质粒的构建及碱性蛋白酶的表达  

Construction of Escherichia coli-Bacillus subtilis Shuttle Vector and Expression of Alkaline Protease

作　　者：梁晓梅[1] 黎明[1] 成堃[1] 贾红红[1] 路福平[1] 

机构地区：[1]天津科技大学生物工程学院教育部工业微生物重点实验室工业酶国家工程实验室,天津300457

出　　处：《生物技术通报》2011年第6期165-169,共5页Biotechnology Bulletin

基　　金：国家"863"计划项目(2007AA02Z212)

摘　　要：构建了一个可以在大肠杆菌和枯草芽孢杆菌中复制,并可以在枯草芽孢杆菌中表达外源蛋白的穿梭表达载体pBE2R。该穿梭表达载体是以pBE2为基本骨架,引入来自pWB980质粒的P43强启动子,并在其下游引入嗜碱芽孢杆菌碱性蛋白酶信号肽和前肽构建而成。试验结果表明,该穿梭表达载体pBE2R可以在大肠杆DH5α和枯草芽孢杆菌WB600中稳定存在,并可以使外源蛋白在枯草芽孢杆菌进行分泌表达,同时也为碱性蛋白酶定向改造提供了高通量筛选平台。A new Escherichia coli-BaciUus subtilis shuttle vector pBE2R was constructed to replicate itself in both hosts and express the foreign protein effectively in B. subtilis. The P43 promoter which was derived from the plasmid pWB980 was introduced into the MCS of the pBE2. Then the fragment of signal peptide and pro-peptide of the alkaline protease derived from the B. alcalophilus was inserted into the downstream of the P43 promoter to produce the new shuttle vector pBE2R. It was demonstrated that the pBE2R could copy itself steadily both in E. coil DH5ot and Bacillus subtilis WB600. The shuttle vector pBE2R not only provided a convenient tool for the expression of the foreign protein in B. subtilis, but also established a platform for the high-throughput screening of the alkaline protease in the process of the directed evolution of the alkaline protease.

关 键 词：穿梭载体 P43启动子 碱性蛋白酶 信号肽 前肽 

分 类 号：Q78[生物学—分子生物学]