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Virologica Sinica, 22 (1) : 8-13, 2007
Research Article
Construction and Characterization of Hepatitis B Virus Infectious Replicon
1Departmant of Virology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, P. R. China 2Division of Clinical Immunology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, P. R. China 3Institute of Virology, Duisburg-Essen University, Essen 45147, Germany
To establish a replication cellular model of hepatitis B virus (HBV) and determine its application in antiviral drug evaluation, we constructed an expression plasmid which contained 1.3 copies of the HBV genome, and measured the level of viral replication after transient transfection in Huh7 cells. We then observed the effect of antiviral drug administration. 1.3 fold of the HBV(ayw) gene fragment was cloned into pCR2.1 by PCR and restriction endonuclease digestion. The recombinant plasmid was transient transfected into Huh7 cells, HBsAg,HBeAg and HBV DNA in supernatant of Huh7 cells were measured by ELISA and real-time PCR respectively; intracellular HBV replicative intermediates and intracellular HBV transcripts were detected by Southern blot and Northern blot respectively. The antiviral effect of adefovir, a novel
anti-HBV nucleotide analogue, was evaluated in this cellular model system. The results indicated that a recombinant plasmid of HBV replicon was constructed successfully; the HBV genome carried in plasmid pHBV1.3 could efficiently replicate and be expressed in Huh 7 cells, adefovir could inhibit HBV replication in this cellular model, and the inhibition was dosage-dependent. The conclusion is HBV replicon, which can initiate viral replication efficiently in hepatoma cells, may be a useful tool in the study of HBV replication and antiviral drug.
  Published online: 20 Feb 2007
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