乙型肝炎病毒剪接蛋白在大肠埃希菌中的表达和纯化  

Expression and purification of hepatitis B spliced protein(HBSP) in Escherichia coli

作　　者：吴龙飞[1] 陈金烟[1] 焦伯延[1] 郑大利[1] 林旭[1] 

机构地区：[1]福建医科大学分子医学研究中心,消化道恶性肿瘤教育部重点实验室,福建福州350004

出　　处：《中国病原生物学杂志》2012年第5期321-324,328,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30970163);福建省高校科技创新团队培育计划基金项目(No.FMU-RT001)

摘　　要：目的高效表达并获得高纯度的乙型肝炎病毒剪接蛋白(hepatitis B splicing protein,HBSP)。方法将HBSP编码基因克隆入原核表达载体pET43.1a(+),并在目的基因的C端引入StrepⅡ标签的编码序列,构建HBSP表达载体;将StrepⅡ标签的编码序列置于HBSP基因片段的N端,同时在StrepⅡ和HBSP之间引入终止密码子以构建对照载体。将以上质粒分别转化大肠埃希菌Rosetta(DE3),以IPTG诱导,SDS-PAGE检测融合蛋白的表达及其可溶性;通过Strep-Tactin系统亲和层析纯化蛋白,Western blot检测其反应原性。结果成功构建HBSP重组表达载体pET-43-HBSP-StrepⅡ及对照载体pET-43-StrepⅡ,经IPTG诱导后,分别在大肠埃希菌中高表达Nus-HBSP-StrepⅡ及Nus-StrepⅡ融合蛋白,且融合蛋白主要以可溶形式存在于细菌裂解上清液中。通过亲和层析获得纯度超过95%的Nus-HBSP-StrepⅡ及Nus-StrepⅡ融合蛋白,两融合蛋白均能被Strep.TagⅡ单抗识别。结论在大肠埃希菌中可高效、可溶性表达并获得高纯度的HBSP融合蛋白及对照蛋白,为HBSP功能研究打下良好基础。Objective To obtain a large amount of highly pure hepatitis B spliced protein（HBSP）.Methods The HBSP gene fused with StrepⅡ tag sequences at the C-terminus was cloned into the prokaryotic expression vector pET43.1a（＋） to generate an HBSP expression recombinant vector.The HBSP gene fused with StrepⅡ tag sequences at the N-terminus with a stop codon was cloned into pET43.1a（＋） to generate a control plasmid.The recombinant plasmid was transformed into Escherichia.coli.Rosetta（DE3） separately and induced with IPTG.The recombinant proteins were assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） and purified with Strep-Tactin affinity chromatography.Results The recombinant plasmid pET-43-HBSP-StrepⅡ and control plasmid pET-43-StrepⅡ were successfully constructed.With induction by IPTG,they respectively expressed the fusion proteins Nus-HBSP-StrepⅡ and Nus-StrepⅡ at a high level.The expressed proteins were mainly present in lysate supernatants.HBSP fusion proteins with a purity greater than 95% were obtained by Strep-Tactin affinity chromatography.Conclusion HBSP fusion proteins and control proteins that were highly soluble and pure were prepared in an E.coli system,laying a foundation for further functional studies.

关 键 词：乙型肝炎病毒 RNA剪接 大肠埃希菌 基因表达 

分 类 号：R373.21[医药卫生—病原生物学] R378.21[医药卫生—基础医学]