枯草杆菌表达载体pUB23的构建及地衣杆菌α-淀粉酶基因的表达  

Construction of pUB23 Vector for Expression of B.Licheniformis α-Amylase Gene in B.Subtilis

作　　者：罗进贤[1] 李文清[1] 王红革[1] 朱碧瑜 

机构地区：[1]中山大学生物系及生物工程研究中心,广州510275

出　　处：《生物化学杂志》1993年第2期194-199,共6页

基　　金：国家"七五"重点科技攻关项目(75-71-01-02)

摘　　要：将克隆的解淀粉芽胞杆菌强启动子经DNA序列分析后连接到能在枯草杆菌中复制的质粒pUB18上,构建枯草杆菌表达载体pUB23。为了测试构建的表达载体能否表达外源基因,将地衣杆菌抉失了启动子的α-淀粉酶基因接到pUB23上启动子的下游,组建重组质粒,转化枯草杆菌QB1130(amy^-),获得能分泌α-淀粉酶的转化株,证明缺失了启动子的结构基因在pUB23上克隆启动子的启动下获得表达。酶活力测定结果表明,表达水平是用原启动子时的2.5倍.The cloned 0.8kb promoter fragment of B. amyloliquefaciens was sequenced by Sanger's dideoxy chain termination method and used to construct the expression vector for B. subtilis. The sequence data showed that the cloned fragment contained two tamdenly aligned promoters. To construct the expression vector, the cloned fragment was excised from plasmid pAED23 by EcoRⅠ-HindⅢ digestion and ligated to EcoRⅠ, HindⅢ sites of pUB18 which is able to replicate in B.subtilis. The resulted plasmid was designated as pUB23. The function of the constructed vector,was then tested by inserting a 1.7 kb fragment containing the B. licheniformis α-amylase gene lacking the promoter into pUB23 downstream of the cloned promoter to yield plasmid pUBA23 for expression in B.subtilis QB1130(amy^-).Amylase positive clonies were obtained, indicating the expression of the amylase structural gene in B.subtilis was under the control of cloned promoter in pUB23. The increase in expression level of amylase activity was about 2.5 times the original.

关 键 词：枯草杆菌 表达载体 地衣杆菌 

分 类 号：Q81[生物学—生物工程]