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Article in Chinese | WPRIM | ID: wpr-332586


<p><b>OBJECTIVE</b>To construct a lamivudine-resistant plasmid containing 1.2 unit genome of duck hepatitis B virus and identify its replication and drug-resistance in avian LMH hepatica cells.</p><p><b>METHODS</b>The recombinant plasmid PBS-DHBV1.2 was constructed using the 1.2-genome length DHBV DNA sequence from a dimer DHBV genome with pcDNA3.1 as the template. With site-directed mutagenesis, we obtained PBS-DHBV1.2-M512V plasmids with polymerase gene mutation from PBS-DHBV1.2. Two constructed plasmids were transiently transfected into LMH cells using FuGENETM6 transfection reagent and cultured in the medium containing different concentrations of lamivudine. Southern blot hybridization was performed to detect DHBV replication intermediates.</p><p><b>RESULTS</b>PCR amplification, restriction digestion and plasmid sequencing all confirmed successful construction of PBS-DHBV1.2-M512V recombinant plasmid. Southern blot analysis identified the presence of all the expected DHBV replication intermediates in LMH cells. The replication capacity of the mutant plasmid was decreased by 2.7 times compared with that of the wild plasmid. The IC(50) of lamivudine was 37.12∓8.81 ng/ml for the mutant, greater than that of the wild plasmid (10.90∓4.80 ng/ml).</p><p><b>CONCLUSION</b>Compared with the wild plasmid, the mutant plasmid has a lower replication capacity and sensitivity to lamivudine in vitro.</p>

Antiviral Agents , Pharmacology , Drug Resistance, Viral , Genetics , Hepatitis B Virus, Duck , Genetics , Lamivudine , Pharmacology , Mutagenesis, Site-Directed , Plasmids
Article in Chinese | WPRIM | ID: wpr-233695


<p><b>OBJECTIVE</b>To study hepatitis B virus (HBV) expression in 3 hepatocytes infected with recombinant adenovirus containing 1.2-copy HBV DNA.a</p><p><b>METHODS</b>A chicken hepatoma cell line and two human hepatocytes were infected by the recombinant adenovirus containing 1.2-copy HBV DNA at 25 pfu/cell. HBV-specific mRNA was detected by RT-PCR 3 days after the infection, and HBsAg and HBeAg were detected by ELISA and HBV DNA by real-time PCR daily after the infection.</p><p><b>RESULTS</b>HBV mRNA expression was detected in all the 3 cells after recombinant adenovirus infection, and the quantities of HBV DNA and HBV antigens in the culture supernatant increased with the passage of time.a</p><p><b>CONCLUSION</b>Infection with the recombinant adenovirus containing 1.2-copy HBV DNA can mediate HBV infection in the 3 cells in vitro.</p>

Adenoviridae , Genetics , Animals , Cell Line, Tumor , Culture Media, Conditioned , Metabolism , DNA, Recombinant , Genetics , DNA, Viral , Genetics , Metabolism , Gene Expression , Hepatitis B Antigens , Metabolism , Hepatitis B virus , Hepatocytes , Metabolism , Virology , Humans , Reverse Transcriptase Polymerase Chain Reaction
Article in Chinese | WPRIM | ID: wpr-283126


<p><b>OBJECTIVE</b>o study the replication of hepatitis B virus (HBV) in HepG2 cells infected with Ad-1.2 HBV.</p><p><b>METHODS</b>HepG2 cells were transfected with adenovirus containing 1.2 copies of HBV DNA. The expression of HBV antigens were detected in the culture medium by means of enzyme-linked immunosorbent assay (ELISA), and the covalently closed circular DNA (cccDNA) in the cells was extracted with plasmid extraction kit and detected by real-time PCR with selective primer after treatment with mung bean nuclease.</p><p><b>RESULTS</b>HBsAg, HBeAg and HBV cccDNA were all detected in HepG2 cells after tranfection with Ad-1.2 HBV. HBV cccDNA was detected 1 day after the infection, reaching the peak level 4 days after infection.</p><p><b>CONCLUSION</b>Ad-1.2 HBV-infected cells can serve as the model for screening and evaluation of antiviral agents.</p>

Adenoviridae , Genetics , Calibration , Cell Line, Tumor , DNA, Complementary , Genetics , Metabolism , DNA, Viral , Genetics , Metabolism , Hepatitis B Surface Antigens , Metabolism , Hepatitis B e Antigens , Metabolism , Hepatitis B virus , Genetics , Allergy and Immunology , Metabolism , Physiology , Humans , Polymerase Chain Reaction , Time Factors , Transfection , Virus Replication