分泌载体pUS186的构建及地衣杆菌α－淀粉酶基因在枯草杆菌中的表达和分泌  

Construction of Secretion Vector pUS186 and the Expression of B. licheniformis Amylase Gene in and Product Secretion from B. subtilis

作　　者：李文清[1] 罗进贤[1] 王红革[1] 张添元[1] 

机构地区：[1]中山大学生物工程研究中心及生物系

出　　处：《Acta Genetica Sinica》1994年第4期330-336,共7页

基　　金：国家"七五"重点科技攻关项目

摘　　要：利用枯草杆菌的分泌系统构建分泌型表达载体表达和分泌外源基因产物具有重要的商业价值。我们用鸟枪法克隆了枯草杆菌染色体的启动子和信号肽序列，将克隆的序列连接到能在枯草杆菌中复制的质粒ｐＵＢ１８上，获得分泌型表达载体ｐＵＳ１８６。为了测试构建的载体ｐＵＳ１８６的功能，将地衣杆菌α－淀粉酶基因的缺失了启动子和信号肽序列的片段重组进该质粒，经过Ｂａｌ３１酶切，Ｔ４ＤＮＡ聚合酶补齐等处理，获得ｐＵＳＡ１８６Ⅱ及ｐＵＳＡ１８６Ⅰ系列质粒，将这些重组质粒转化枯草杆菌ＱＢ１１３０（ａｍｙ－）后都能向胞外分泌淀粉酶，酶活测定结果表明，基因表达水平比用原有的启动子高１－２倍，蛋白质分泌率在８４－９６％之间。secretive expression vector pUS186 was constructed by ligating the cloned promoter-signal sequence coding region from B. subtilis chromosome to plasmid pUB18 which replicates in B. subtilis. To test the function of the constructed vector, the B. licheniformis α-amylase gene lacking the promoter and signal sequence was introduced downstream of the cloned signal sequence fragment on pUS186 resulting in plasmid pUSA186 which was treated with T4 DNA polymerase and Bal31 and T4 DNA polymerase together to readjust the reading frame. The resultant plasmids pUSA186II and pUSA186I series were obtained and introduced into B. subtilis QB1130(amy-),Most of the transformants secrete amylase into the medium. The secretion rate is 84-96%.

关 键 词：枯草杆菌 信号肽 序列 分泌载体 

分 类 号：Q782[生物学—分子生物学]