海洋创伤弧菌反式翻译系统关键因子SmpB基因的克隆及原核表达

创伤弧菌(Vibrio vulnificus)是一种重要的"人鱼共患病"病原菌。通过克隆创伤弧菌反式翻译系统核心因子小蛋白B(Small molecular protein B,SmpB)基因,构建携带目的基因的原核表达质粒,为后续研究SmpB蛋白的互作网络、SmpB蛋白与创伤弧菌致病性之间的关系并以此开发新型的抑菌靶标奠定基础。使用LiCl沉淀法提取创伤弧菌基因组DNA,以它为模板,PCR扩增目的基因,并构建到pET-28a原核表达载体上测序鉴定后对SmpB序列进行生物信息学分析,将正确的重组质粒转化E.coli BL21(DE3),IPTG诱导表达,SDS-PAGE凝胶电泳鉴定。结果表明使用LiCl沉淀法成功提取到高质量创伤弧菌基因组DNA,以其为模板,扩增到smpB基因,并成功构建pET-28a原核表达重组质粒,测序鉴定正确;smpB基因全长为486 bp,编码161个氨基酸,分子量为18.41 kD,理论等电点为10.28,不稳定系数为35.02,总平均亲水性为-0.635,SmpB蛋白整体表现为稳定亲水性蛋白。生物信息学分析显示其高级结构核心部分为5个β折叠组成的桶状结构,外围由3个α螺旋组成,SmpB C-端亦为α螺旋。诱导表达的重组融合蛋白相对分子质量大小在25.0 kD附近,显示在E.coli中成功表达了SmpB蛋白。

Construction and Expression of Prokaryotic Expression Vector for SmpB of a Key Factor in Trans-translation System of Vibrio vulnificus

Vibrio vulnificus is an important pathogen infecting both aquatic animals and human.To clone the small molecular protein B(SmpB)gene of the key factor of the trans-translation system in V.vulnificus and to construct the prokaryotic expression plasmid carrying the target gene would lay the foundation for the subsequent research on the interaction network of SmpB,and its relationship with the pathogenicity of V.vulnificus,as well as for development of new anti-bacterial target.The genomic DNA of V.vulnificus was extracted by LiCl sedimentation method,and the target gene was amplified by PCR,and constructed into a prokaryotic expression vector of pET-28a.The constructed recombinant plasmid pET-28a-SmpB was identified by PCR and sequencing,then the bioinformatics analysis of SmpB protein was performed.Further the correct recombinant plasmid was transformed into Escherichia coli BL21(DE3)for expression under induction of IPTG at low temperature,and the expressed product was identified by SDS-PAGE gel electrophoresis.The results showed that the high-quality V.vulnificus genomic DNA was successfully extracted by LiCl sedimentation method,which was used as a template for specific amplification of the smpB,the pET-28a-SmpB was successfully constructed,and verification by sequencing was correct.The full length of smpB gene was 486 bp and encoded 161 amino acids,the molecular weight was 18.41 kD,the theoretical isoelectric point was 10.28,the instability coefficient was 35.02,the total average hydrophilicity was-0.635,and SmpB protein was stable hydrophilic protein as a whole.The core of SmpB three-dimensional structure was composed of 5 strands of the β-barrel,the periphery was composed of 3 α-helix,and the C-terminal of SmpB protein was an α-helix.The relative molecular weight of the recombinant fusion protein was around 25.0 kD,indicating the SmpB protein was successfully expressed in E.coli.