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- W2393426913 abstract "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 confocal laser scanning microscopy (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 was 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-GRP78 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 pcDNA3.1 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 pcDNA3.1-GRP78 may create a transient GRP78 over-expression,providing a possible tool for future research on GRP78." @default.
- W2393426913 created "2016-06-24" @default.
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- W2393426913 date "2009-01-01" @default.
- W2393426913 modified "2023-09-24" @default.
- W2393426913 title "Subcellular localization of GRP78 in HepG2 and construction of its eukaryotic vector" @default.
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