Objective       To construct the HBx-defective mutant plasmid pUC-HBV1.0.X7

and investigate the effect of the absence of HBx on HBV replication and transcription.       Methods       The wild-type plasmid pUC-HBV1.0 which containing full-length HBV genome was used as a template

and the plasmid pUC-HBV1.0.X7 with a stop codon at amino acid 7 of HBx was constructed by site-directed mutagenesis

which was then identified by HindⅢ and SacⅠ  restriction digestion and DNA sequencing. Then the plasmids pUC-HBV1.0 and pUC-HBV1.0.X7 we

re both digested by SapⅠ enzyme to obtain linear HBV DNA

and the two kinds of linear HBV DNA were separately transfected into HepG2 cells with PolyJet

TM

Reagent. The expression of HBx was detected by Western blotting. Cytoplasmic HBV DNA and cccDNA were both detected by Southern blotting and real-time PCR. The transcription of HBV pgRNA was quantified by real-time PCR at different time points after .transfection.       Results       The HBx-deficient plasmid pUC-HBV1.0.X7 was verified to be constructed successfully by double digestion and DNA sequence analysis. The expression of HBx was detected in the group of wild-type

while it was below the detection limit by Western blotting in the HBx mutant. The levels of cccDNA formation were not significantly different in the wide type HBx and HBx mutant (P0.05). The levels of cytoplasmic HBV DNA and pgRNA in the HBx mutant were reduced by 50% to 70% respectively compared to those in the wild type HBx at 96 h (P0.05).       Conclusion       Although the absence of HBx does not affect cccDNA formation

it can downregulate HBV replication and transcription.