溶血素 E基因原核表达载体的构建与蛋白诱导表达  

Construction of A Prokaryotic Expression Vector for the Hemolysin E Gene and Induction of Protein Expression

作　　者：何学仙[1] 何晓艳 潘晓玥 HE Xuexian;HE Xiaoyan;PAN Xiaoyue(Cerebrospinal Fluid Laboratory of Neurology Department,General Hospital of Ningxia Medical University,First Clinical Medicine College of Ningxia Medical University,Yinchuan 750004,China;Department of Quality Control,Ningxia Minkang Hospital,Yinchuan 750011,China;School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学总医院神经内科脑脊液检测室,宁夏医科大学第一临床医学院,银川750004 [2]宁夏民康医院质量控制科,银川750011 [3]宁夏医科大学基础医学院,银川750004

出　　处：《宁夏医科大学学报》2024年第7期663-666,共4页Journal of Ningxia Medical University

基　　金：宁夏自然科学基金项目(2020AAC03375)。

摘　　要：目的构建大肠杆菌(Escherichia Coli,E.coli)溶血素E(Hemolysin E)基因的重组质粒pCZN1-hlyE,诱导表达目的蛋白。方法通过NCBI数据库检索获取E.coli Hemolysin E基因序列(NC_000913.3),采用基于PAS(PCR-based Accurate Synthesis)的全基因合成法,设计全长拼接引物,合成目的基因,并成功构建重组质粒pCZN1-hlyE。将构建好的pCZN1-hlyE重组质粒转入大肠杆菌TOP10菌株中,并用异丙基-β-D-硫代半乳糖苷(IPTG)诱导Hemolysin E基因的表达,生成Hemolysin E毒力蛋白。结果经SDS-PAGE凝胶电泳,Western blot分析鉴定目的蛋白Hemolysin E分子量为28.72 kDa,与Hemolysin E基因核酸序列经DNAMAN软件分析所得蛋白分子量一致。结论被诱导表达的Hemolysin E蛋白存在于菌体裂解液上清中,并纯化制备重组蛋白。Objective To construct an expression vector for the Hemolysin E gene of Escherichia Coli,to induce the expression of the target protein in order to lay the foundation for the regulation of Hemolysin E expression by genetic engineering and the development of clinical diagnostic immunoreagents.Methods The E.coli Hemolysin E gene sequence(NC_000913.3)retrieved from NCBI database was synthesized through the PCR-based Accurate Synthesis(PAS)method.The full-length splicing primers were designed to synthesize target genes and the recombinant plasmid pCZN1-hlyE was successfully constructed.The constructed pCZN1-hlyE recombinant plasmid was transferred into E.coli TOP10.After identification,the expression of Hemilysin E gene was induced with propyl-β-D-thiogalactoside(IPTG)to generate the Hemolysin E virulence protein.Results By SDS-PAGE gel electrophoresis and Western blot analysis,the molecular weight of the target protein Hemolysin E was 28.72 kDa,which was consistent with the molecular weight of the protein obtained by DNAMAN software analysis of the nucleic acid sequence of the Hemolysin E gene.Conclusion The induced expression of Hemolysin E protein is present in the supernatant of bacterial lysate and purified to prepare recombinant protein.

关 键 词：溶血素E 全基因合成 原核表达 蛋白诱导表达 

分 类 号：Q786[生物学—分子生物学]