大肠杆菌-枯草杆菌穿梭载体的构建及应用

以大肠杆菌pMB1复制类型质粒pUC18为模板,PCR扩增了700bp的含质粒复制起始点和复制蛋白序列的DNA片段,克隆到质粒pBluskm的多克隆位点,得到含大肠杆菌质粒复制基因的中间载体pE3;以革兰氏阳性菌质粒pGDV1为模板,PCR扩增了含革兰氏阳性菌质粒正负链复制区域的2·5kb片段,并与pE3载体上的大肠杆菌复制子和复制蛋白连接构建了3·2kb的大肠杆菌-枯草杆菌穿梭载体pGJ103。电转化pGJ103质粒至枯草杆菌DB104和1A747,均能稳定地复制。以pGJ103为骨架,克隆了不同大小的生物素基因片段,均能正确稳定地复制,证明载体有良好的承载能力。将2kb的热稳定性β-半乳糖苷酶基因(bga)克隆至pGJ103构建了载体启动子检测载体pGJ199。用pGJ199质粒载体作为启动子检测载体,对革兰氏阳性菌启动子片段P59进行了转录活性检测。并以该载体为启动子探测工具,通过鸟枪法得到了大量启动子片段。

Construction and characterization of a versatile E. Coli- B. Subtilis shuttle vector

In this work, a 700 bp pMB1 replicon of Escherichia coli was cloned into pBluskm, yielding pE3. While a 2.5 kb fragment, containing single-strand and double-strand replication function in B.subtilis, was amplified from plasmid pGDV1 and ligated with the pMB1 replicon, yielding E.coli-B.subtilis shuttle vector pGJ103. This shuttle vector (3.2 kb) can replicate steadily in E.coli DH5a and B.subtilis. In another experiment, different sizes of DNA fragments were cloned into pGJ103 to determine stability of replication. Result showed the shuttle vector could still replicate stably when 5 kb foreign DNA was cloned. The versatile and flexible usage of the shuttle vector constructed in this study was demonstrated by construction of promoter probe vector and determination of many promoters.