2004•Chinese Journal of Current Advances in General SurgeryRequires access
Construction of HBV gene eukaryotic expression vector containing 3.0-fold-overlength genome of HBV and its expression
Xiaohong Liang
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Abstract
Objective: To construct HBV gene eukaryotic expression vector containing 3 0-fold-overlength genome of HBV and HBV transfected cell model.Methods: 9.6kb fragment of HBV genome,derived from pUC19-3HBV,was cloned into EcoRⅠ and HindⅢ sites of the eukaryotic expression vector pcDNA_3 to construct the recombinant plasmid pcDNA_3-3HBV.Human hepatoma cell HepG2 was transfected with pcDNA_3-3HBV and screened with antibiotic G418.HBeAg and HBsAg were measured by ELISA kits.HBpreS2 and HBX mRNA expression were tested by RT-PCR.Results: The expression vector pcDNA_3-3HBV was constructed successfully.After stable transfection,HepG2.3Z was established with G418 screening.HepG2.3Z cells could express HBsAg and HBeAg stably.HBpreS2 and HBX mRNA were also measured by RT-PCR.Conclusion:pcDNA_3-3HBV could initiate viral replication efficiently in infected cells.HepG2.3Z is expected to be a novel tool for investigating HBV replication in vitro.[