深海来源低温诱导表达载体pSW4的构建及其应用

Construction of cold inducible expression vector pSW4 and expression of DNA polymerase III epsilon subunit

摘要:

【目的】在深海来源穿梭载体pSW2的基础上构建低温诱导高效表达载体，为最终获得环境污染物高效降解菌提供基础和工具。【方法】通过最小化敲除实验确定载体必需基因，以绿色荧光蛋白GFP为报告基因检测载体的表达能力变化，进一步添加纯化标签、多克隆位点及替换启动子等改造获得低温诱导表达载体pSW4。【结果】敲除实验结果显示编码单链结合蛋白的基因fpsB敲除后载体的表达效率显著提高。以GFP为报告基因检测发现，pSW4的表达效率较pSW2有显著提升(4 °C条件下提高10.7倍)。以深海细菌Shewanella piezotolerans WP3为宿主菌，应用pSW4为表达载体表达深海细菌Shewanella psychrophila WP2的聚合酶亚基，检测显示其在Mg2+条件下具有切割单链DNA的核酸酶活性，而在Mg2+或Mn2+条件下具有切割双链DNA的核酸酶活性。【结论】构建了低温诱导表达载体pSW4，并证实了其适用性，有助于今后构建环境修复菌及相关的应用性研究。

Abstract:

[Objective] Deep-sea derived shutter vector pSW2 has been constructed previously. In this study, a cold inducible expression vector was constructed based on pSW2 to provide useful explant materials and toolbox for the construction of an engineered strain that can degrade environmental pollutants. [Methods] Mutation experiments were performed to identify essential gene in the vector, and green fluorescent protein (GFP) was used as reporter gene to test the expression efficiency of mutated vectors. Finally, the cold inducible vector pSW4 was constructed by adding purification tags and multiple cloning sites, and by replacing of the promoter region. [Results] The mutation experiment indicated that the GFP intensity was significantly increased by deletion of fpsB gene, which encodes the ssDNA binding protein. The expression efficiency of pSW4 was higher than that of pSW2 (10.7-fold at 4 °C) by using GFP as reporter gene. The sps01203 gene in the deep-sea bacterium Shewanella psychrophila WP2 was successfully expressed using Shewanella piezotolerans WP3 as expression host and pSW4 as expression vector. Subsequent assay indicated that the protein possesses nuclease activity. Specifically, Mg2+ is essential for the ssDNA substrate, whereas Mg2+ or Mn2+ is required for the dsDNA substrate. [Conclusion] A cold-induced expression vector pSW4 has been successfully constructed, and its applicability has been verified. Thus, our study will contribute to the further applied research of bioremediation bacterium.