[Construction of pEGFP-HBx gene recombinant plasmid and establishment of stable transfected HBx gene liver oval cell strain].
OBJECTIVE
To construct pEGFP-HBx eukaryotic expression plasmid and establish stable and effective transfected rat oval cell (LE/6) strain expressing EGFP-HBx fusion protein to explore the roles of HBx gene and oval cell in carcinogenesis of hepatocellular carcinoma (HCC).


METHODS
HBx gene with EcoRI and Hind III endo-enzyme sites was obtained by using PCR from plasmid pcDNA3.1-HBx. The purified HBx gene fragment was inserted into pEGFP-N1 expression vector, and the recombinant plasmid pEGFP-HBx was identified by restriction endonuclease and DNA sequencing analysis. LE/6 cells were transfected with recombinant pEGFP-HBx by lipofectamine reagent. Resistant to G418 clones were selected, and expression of EGFP-HBx fusion protein in clones were examined directly with fluorescence microscope, and these clones were isolated and proliferated. The expression of HBx was detected by RT-PCR analysis and immunocytochemistry.


RESULTS
Plasmid pEGFP-HBx has whole HBx gene base and correct reading frame as indicated by restriction endonuclease and DNA sequencing analysis. After transfecting with pEGFP-HBx plasmid, LE/6 cell clones expressing EGFP-HBx fusion protein were obtained. RT-PCR analysis and immunocytochemistry showed that HBx gene was only expression in transfected pEGFP-HBx cells.


CONCLUSIONS
The pEGFP-HBx recombinant expression vector was successfully constructed, and the stable transfected LE/6 strain expressing EGFP-HBx fusion protein was successfully established. It will be helpful in the further study on the roles of HBx and liver oval cell in carcinogenesis of HCC.
Li
Chang-hai
CH
Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Chen
Xiao-ping
XP
Liang
Hui-fang
HF
Wang
Heng-yi
HY
Wang
Yan-jun
YJ
Dong
Wei
W
D000818:Animals; D002460:Cell Line; D005822:Genetic Vectors; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D051381:Rats; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics*; D014162:Transfection
D015534:Trans-Activators