极端嗜盐古菌质粒pSCM201衍生穿梭表达载体的构建及应用

Construction and application of a novel shuttle expression vector based on haloarchaeal plasmid pSCM201

摘要:

摘要：【目的】利用有自主知识产权的嗜盐古菌θ型复制质粒和启动子，构建在极端嗜盐古菌模式菌株西班牙盐盒菌（Haloarcula hispanica）中方便使用、功能完善的基因表达载体。【方法】以pSCM201的最小复制子为基础，通过引入莫维诺林抗性基因，大肠杆菌质粒复制子以及氨苄抗性基因，构建了一个新的嗜盐古菌-大肠杆菌穿梭载体。利用定点突变和末端补平法依次将其中多余的酶切位点去除后，再添加hsp5启动子核心序列、人工合成的多克隆位点以及蛋白纯化标签His?Tag等重要元件成功构建了嗜盐古菌表达载体pSCM307。将报告基因bgaH插入到该载体的多克隆位点中并转化H. hispanica AS2049，通过X-gal平板筛选和β-半乳糖苷酶酶活实验检测pSCM307的表达能力。【结果】pSCM307具有独立的自主复制能力，其多克隆位点方便实用，报告基因bgaH在hsp5启动子控制下实现了高效表达。【结论】成功构建了嗜盐古菌领域中第一个方便使用的基因表达载体。

Abstract:

Abstract: [Objective] pSCM201 is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea. For gene expression and function analysis of specific genes in model strain Haloarcula hispanica, a novel shuttle expression vector based on pSCM201 was developed. [Methods] By combining the minimal replicon of pSCM201, the pUC19 replicon of Escherichia coli, and two antibiotic genes, a new shuttle vector between haloarchaea and E. coli was established. Furthermore, the hsp5 core promoter, multiple clone sites and a C-terminal His?Tag sequence were added to construct the expression vector pSCM307. With the help of reporter gene bgaH, the replication ability and gene expression of pSCM307 were tested in H. hispanica AS2049 by PCR identification and β-galactosidase activity assay. [Results] pSCM307 could stably self-replicate in AS2049, and bgaH was successfully expressed under the control of hsp5 promoter. [Conclusions] A user-friendly expression vector was successfully constructed in haloarchaea.