CAJ | 학술논문

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-gamiTM2(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HBeAg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.
목적 관찰단백전도역(PTD)-HBcAg융합단백적세포막천투작용.방법 합성전록반식격활단백(Tat)-PTD편마서렬,PCR기술확증HBcAg전장기인,통과중첩연신PCR편단병접법장Tat-PTD편마서렬화HBcAg전장기인융합,장융합기인극륭도pMAL-c2X원핵표체재체중.도선측서정학적구건질립,전화대장애희균Rosetta-gamiTM2(DE3),이병기-β-D류대반유당감유도융합단백표체,Western인적감정,친화층석순화융합단백PTD-HBcAg,동양방법득도HBcAg단백작위대조.순화단백가입세포배양개질중,간접면역형광검측진입세포내적PTD-HBcAg화HBcAg.결과 대장애희균중과도표체적융합단백가통과친화층석순화,Western인적증실순화적PTD- HBcAg화HBcAg능피HBeAg단극륭항체식별,세포면역형광검측증실PTD-HBcAg가과막전도입세포내,이단독적HBcAg미능재세포내검측도.결론 융합단백PTD-HBcAg가재원핵표체계통중고효표체、분리순화,PTD능개도HBcAg천투세포막진입세포.
0bjective To observe the cell membrane penetration of protein transduction domain (PTD)-HBeAg 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 eontr0l.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 HBeAg monoclonal antibody in Western blot analysis.IFA visualization showed that PTD-HBeAg could be introduced into HUH-7 ceils 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 eell membrane into the cells.