Trans-activator of transcription protein transduction domain mediated cell membrane penetration of HBcAg
- VernacularTitle:转录反式激活蛋白转导域介导HBcAg穿透细胞膜的作用
- Author:
Qingchun PAN
;
Guoqing ZANG
;
Yongsheng YU
;
Zhenghao TANG
;
Wei ZHANG
;
Jingchao HAN
- Publication Type:Journal Article
- Keywords:
Trans-activators;
Transduction,genetic;
Hepatitis B core antigens;
Cell membrane
- From:
Chinese Journal of Infectious Diseases
2008;26(6):336-340
- CountryChina
- Language:Chinese
-
Abstract:
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.
