GRP78在HepG2细胞的亚细胞定位及真核表达

目的 构建GRP78的真核表达载体以检测GRP78在HEK293细胞中的表达和定位.方法采用激光共聚焦显微镜观察GILP78在HepG2细胞中的定位,用RT-PCR方法从HepG2细胞中扩增GRP78 cDNA序列,构建真核表达载体peDNA3.1-GRP78,转染HEK293细胞,分别用Western blotting和激光共聚焦显微镜检测GRP78在HEK293细胞中的表达和定位.结果 GRP78在HepG2细胞的胞膜和胞质均有表达,GRP78在细胞膜上均匀分布.双酶切鉴定和核酸序列分析证实pcDNA3.1-GRP78真核表达质粒构建成功,该表达质粒转染HEK293细胞后可以瞬时表达GRP78蛋白.结论 正常状态下,GRP78在HepG2细胞胞膜和胞质均有表达.pcDNA3.1-GRP78转染HEK293细胞后能瞬时表达GRP78蛋白,为进一步研究GRP78的功能提供了重要工具.

Subcellular localization of GRP78 in HepG2 and construction of its eukaryotic vector

Objective Previous study had shown that GRP78 could specifically bind to the preS1 region of HBV large surface protein, indicating its important role in mediating HBV entry into hepatocytes. Aim of present study was to investigate the subcellular distribution of GRP78 in HepG2 cells and construct a eukaryotic vector for GRP78,to provide preliminary data for future study in vitro. Methods The subcellular localization of GRP78 in HepG2 cells was observed with corffocal laser scanning mieroscopy (CLSM).The full-length cDNA of GRP78 was obtained from HepG2 cells by RT-PCR and linked to eukaryotic expression vector pcDNA3.1(+) to obtain the recombinant plasmid pcDNA3.1-GRP78,which was identified by Hind Ⅲ/BamH Ⅰ double digestion and sequencing.Finally,the recombinant plasmid wets transfected into HEK293 cells by Liofectamine 2000.The expression and position of GRP78 in HEK293 were detected by Western blotting and CLSM Results In normal conditions,GRP78 was expressed both on cell membrane and cytoplasm of HepG2 cells.Recombinant plasmid pcDNA3.1-GRF78 was proved to be successfully constructed by restriction enzyme digestion analysis with Hind Ⅲ,BamH Ⅰ and DNA sequencing, and the sequence of target gene was completely correct.Western blotting showed that the protein level of GRP78 in pcDNA3.1-GRP78 transfecion group was higher than that in untransfection group and peDNA3.1 transfecion group.The data proved that GRP78 was overexpressed in pcDNA3.1-GRP78-transfected HEK293 cells in protein level.Conclusion In the normal state, GRP78 may be expressed by HepG2 cells in both membrane and plasma with a uniform distribution in membrane. Eukaryotic expression plasmid pcDNA3.1-GRP78 has been successfully constructed.HEK293 cells transfected with peDNA3.1-GRP78 may create a transient GRP8 over-expression,providing a possible tool for future research on GRP78.