ABSTRACT
<p><b>OBJECTIVE</b>To establish a method for efficient transfer of 1.3-fold HBV/C genome into HepG2 cell line using adenoviral vector system for studying the replication and antigen expression of HBV.</p><p><b>METHODS</b>The 1.3-copy overlength genome of HBV genotype C was constructed and cloned into the shuttle vector pAdTrack. After confirmation of the constructed HBV genome by sequencing, the resultant plasmid linearized by digestion with Pme I was transformed into competent E.coli Adeasier-1 cells. The recombinants of pAdEasy-HBV/C were linearized by digestion with Pac I and transfected into the packaging cells (293 cells) via liposome. HepG2 cells were then infected with a proper quantity of the recombinant adenoviruses. The HBV DNA level and HBeAg and HBsAg titers were detected in the cell medium.</p><p><b>RESULTS</b>The 1.3-fold overlength HBV/C genome was efficiently transferred into HepG2 cells via the adenoviral vector system, which resulted in initiation of the virus replication and protein expression in the cells using the viral replication mechanism.</p><p><b>CONCLUSION</b>The adenovirus vector system (AdEasy) allows convenient and effective transfer of HBV genome into HepG2 cells, and provides a convenient means for screening therapeutic drugs against HBV.</p>
Subject(s)
Humans , Adenoviridae , Genetics , Metabolism , Carcinoma, Hepatocellular , Genetics , Virology , Genetic Vectors , Genetics , Genome, Viral , Genetics , Hepatitis B virus , Genetics , Liver Neoplasms , Genetics , Virology , Transduction, Genetic , Tumor Cells, CulturedABSTRACT
<p><b>OBJECTIVE</b>To study the characteristics of the virology background of HLA-A2 restricted HBcAg(18-27) epitope mutations in HBV infected patients in China.</p><p><b>METHOD</b>30 HBV sequences with different genotypes from Genbank were analyzed by bioinformatics and the mismatched primers were designed for constructing a PCR-RFLP method to screen HBcAg(18-27)V/I in China. The distributions of HBcAg(18-27)V/I of 160 samples with HBV genotype B/C infection from 8 areas in China were screened and analyzed by PCR-RFLP and sequencing. The affinity of HBcAg(18-27)V/I to HLA-A0201 was analyzed through referencing the bioinformatics websites.</p><p><b>RESULTS</b>We successfully constructed a PCR-RFLP method for screening HBcAg(18-27)V/I from genotype B/C, and only 3 samples with HBcAg(18-27)V sequence were found in the 160 samples (3/160, 1.88%). The affinity of HBcAg(18-27)I to HLA-A 0201 was lower than the one of HBcAg(18-27)V through bioinformatic analysis (HLA ligand score was 123 vs 156, and the SYFPEITHI score was 22 vs 24).</p><p><b>CONCLUSION</b>The last amino acid of most HBcAg(18-27) sequences of epidemic HBV strains in China is isoleucine, and not valine. Therefore HBcAg(18-27) sequence background in different HBV genotypes should be thoroughly considered when using it as a reference or control in immunological research about HBV.</p>
Subject(s)
Adult , Female , Humans , Male , China , Epidemiology , Computational Biology , DNA, Viral , Genetics , Epitopes, T-Lymphocyte , Allergy and Immunology , Genotype , HLA-A Antigens , Allergy and Immunology , Hepatitis B Core Antigens , Genetics , Allergy and Immunology , Hepatitis B virus , Classification , Allergy and Immunology , Hepatitis B, Chronic , Epidemiology , Allergy and Immunology , Virology , Mutation , Sequence Analysis, DNA , T-Lymphocytes, Cytotoxic , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the distribution of hepatitis B virus (HBV) genotype B subgenotype in China.</p><p><b>METHODS</b>A cohort of 511 patients with chronic HBV genotype B infection, collected from 7 centers across China, were analyzed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or nucleotide sequencing.</p><p><b>RESULTS</b>For the 511 patients, only subgenotype Ba was identified and subgenotype Bj was not found.</p><p><b>CONCLUSION</b>In China, subgenotype Ba was the most prevalent HBV strains, while subgenotype Bj was very rarely found.</p>