转录反式激活蛋白转导域介导HBcAg穿透细胞膜的作用  

Trans-activator of transcription protein transduction domain mediated cell membrane penetration of HBcAg

作　　者：潘庆春[1] 臧国庆[1] 余永胜[1] 汤正好[1] 张韡[1] 韩进超[1] 

机构地区：[1]上海交通大学附属第六人民医院感染科,200233

出　　处：《中华传染病杂志》2008年第6期336-340,共5页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金资助项目（30571669） 志谢 本课题受到上海交通大学医学院附属瑞金医院分子医学中心宋怀东教授和感染科张欣欣教授的大力支持

摘　　要：目的观察蛋白转导域（PTD）-HBcAg融合蛋白的细胞膜穿透作用。方法合成转录反式激活蛋白（Tat）-PTD编码序列，PCR技术扩增HBcAg全长基因，通过重叠延伸PCR片段拼接法将Tat—PTD编码序列和HBcAg全长基因融合，将融合基因克隆到pMAL-c2X原核表达载体中。挑选测序正确的构建质粒，转化大肠埃希菌Rosetta—gami^TM2（DE3），异丙基-β—D-硫代半乳糖苷诱导融合蛋白表达，Western印迹鉴定，亲和层析纯化融合蛋白PTD-HBcAg，同样方法得到HBcAg蛋白作为对照。纯化蛋白加入细胞培养介质中，间接免疫荧光检测进入细胞内的PTD-HBcAg和HBcAg。结果大肠埃希菌中过度表达的融合蛋白可通过亲和层析纯化，Western印迹证实纯化的PTD-HBcAg和HBcAg能被HBcAg单克隆抗体识别，细胞免疫荧光检测证实PTD-HBcAg可跨膜转导入细胞内，而单独的HBcAg未能在细胞内检测到。结论融合蛋白PTD-HBcAg可在原核表达系统中高效表达、分离纯化，PTD能介导HBcAg穿透细胞膜进入细胞。Objective To observe the cell membrane penetration of protein transduction domain （PTD）-HBcAg fusion protein in vitro. Methods The sequence of trans-activator of transcription （Tat）-PTD was synthesized and the whole HBcAg gene was amplified by polymerase chain reaction （PCR）. Overlap extension PCR was employed to fuse Tat-PTD and whole HBcAg gene. Then the fusion gene was cloned into prokaryotic expression vector pMAL-c2X. The correct vector was transformed into E. coli Rosetta-gamiTM 2 （DE3）, and the protein was induced by isopropyl β-D-1- thiogalactopyranoside（IPTG）. Western blot was used to identify the protein. Furthermore,the fusion protein PTD-HBcAg was purified by affinity chromatography. HBcAg protein expressed using the same methods was employed as control. The purified protein was added to HuH-7 cell culture, then the transduction of PTD-HBcAg and HBcAg in cells were detected by indirect immunofluorescence assay （IFA）. Results The fusion protein was effectively expressed in E. coli and purified by affinity chromatography. Both purified PTD-HBcAg and HBcAg could be recognized by HBcAg monoclonal antibody in Western blot analysis. IFA visualization showed that PTD-HBcAg could be introduced into HuH-7 cells while HBcAg only could not be detected in cells. Conclusions PTD-HBcAg fusion protein can be expressed effectively and purified in prokaryotic expression system. PTD could mediate HBcAg penetrating cell membrane into the cells.

关 键 词：反式激活因子类 转导 遗传 肝炎核心抗原 乙型 细胞膜 

分 类 号：R512.62[医药卫生—内科学] R733.71[医药卫生—临床医学]