Abstract:
Objective To establish HBV replication cell models.
Methods Using molecular subclone technique,pcDNA3-3 HBV was constructed by insertion of 9 600 bp fragment of HBV adr subtype genome into the EcoRⅠ and HindⅢ sites of pcDNA3.Hep G2 was transfected with pcDNA3-3 HBV by using lipofectamine transfection reagent and screened with antibiotic G418.
Results The plasmid pcDNA3-3 HBV was constructed successfully.After stable transfection,HBsAg and HBeAg were positive in the supernatant which tested by ELISA.S gene and pre-S gene were also positive which tested by PCR.cccDNA was found in the cells by PCR,and HBV pre2S1 mRNA was measured by RT-PCR.
Conclusion pcDNA3-3 HBV can be expressed,transcribed and replicated in Hep G2 cells.