ABSTRACT
<p><b>OBJECTIVE</b>To construct a stable cell line with permanent secretion of recombinant hepatitis B virus (HBV) vector, which express blasticidin resistant gene.</p><p><b>METHODS</b>Replication-defective HBV vector, pCH-BsdR, which express blasticidin resistance gene was constructed by deleting the HBV genes and inserting the blasticidin resistance gene into the S region. The G418-resistant, the packaging signal deleted HBV helper plasmid, pcDNA3.1-CH3142, and the HBV vector pCH-BsdR were cotransfected into HepG2 cells. Cell clones were selected by the adding of both blasticidin and G418, then serial detection were done.</p><p><b>RESULTS</b>After 36 cell clones were picked and expanded. Three cell clones were defined as the best. Quantity of their HBV DNA were 4.1 x 10(6), 3.6 x 10(6) and 1.2 x 10(6) copies/ml, respectively. Enveloped recombinant, but not wild type HBV were confirmed in the culture medium.</p><p><b>CONCLUSIONS</b>The stable cell lines can realize large preparation of recombinant HBV virions. This will contribute to the use of HBV vector for gene therapy and HBV susceptible cell lines screening.</p>
Subject(s)
Humans , Cell Line , Virology , Clone Cells , Drug Resistance , Gene Expression , Genetic Engineering , Genetic Vectors , Genetics , Metabolism , Hep G2 Cells , Hepatitis B virus , Genetics , Physiology , Nucleosides , Pharmacology , Transfection , Virion , Genetics , Physiology , Virus Assembly , Virus ReplicationABSTRACT
<p><b>OBJECTIVE</b>To characterize the hepatitis A virus (HAV) wild type strains circulating in Hebei Shijiazhuang of China during 2005-2007, to provide the bases for further investigation of the sources of HAV infection.</p><p><b>METHODS</b>The VP1/P2A junction regions were detected by RT-PCR from HAV IgM positives serum samples during 2005 and 2007, the 34 RT-PCR positive samples were sequenced and subjected to phylogenetic analysis by Neighbor Joining (NJ) method.</p><p><b>RESULTS</b>All the detected HAV strains were identified as sub-genotype I A, the homology of nucleotide sequence in the VP1-2A imation region ranged from 95%-100%, the amino acid sequences of HAV strains almost had no difference.</p><p><b>CONCLUSION</b>There are different HAV strains existing in Hebei Shijiazhuang of China, same HAV strain may exist in different areas; or in one area, identical or different HAV strains may be detected. This work provides the bases for further investigation of the sources of HAV infection and also for effectively control measures to prevent the spread of the disease.</p>