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

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.