Construction and expression of HBV gene eukaryotic cell expression vector

Objective:To construct eukaryotic cell expression vector carrying HBV full length gene,and study its replication,transcription and expression in human hepatoma cell line.Methods:pBR322-2HBV and pcDNA3were lined by EcoRⅠdigestion,then HBV full length DNA and linear pcDNA3were extracted.HBV entire genome was inserted at the EcoRⅠsite in the MCS of pcDNA3with T4ligase.Hu-man hepatoma cell HepG2was transfected with pcDNA3-HBV by using lipofectamine transfection reagent and screened with antibiotic G418.HBsAg and HBeAg were measured by ELISA kits.Pre-S1mRNA expres-sion was tested by RT-PCR.Results:We got pcDNA3-HBV expression vector carring HBV full length DNA and identified its sense direction by endonucleases digestion.After transfection,HepG2.02G cell line was established with G418screening and passaged for10months.HepG2.02G cells could express HBsAg constantly.PreS1mRNA was also measured by RT-PCR.Conclusion:pcDNA3-HBV expression vector was constructed,which could be effectively replicated,transcribed and expressed in human hepatoma cell HepG2.And a new cell line HepG2.02G which could express HBV antigen was established with this expression vector.