Cloning and expression HBcAg gene of hepatitis B virus in eukaryotic cells

2. ABSTRACT In the present study, cloning of (HBcAg) gene in Escherichia coli and expression of the gene in eukaryotic cells are reported. Hepatitis B virus (HBV) core antigen gene was amplified from the sera of HBV infected patients in a polymerase chain reaction (PCR) and placed into pUC19 plasmid. The resulting recombinant plasmid, termed pHBc-1, was used in transformation of E. coli cells. The presence of HBcAg in pHBc-1, was confirmed by both PCR and restriction enzyme digestion, which has only single restriction site in HBcAg coding sequence. Later, HBcAg gene was excised from pHBc-1 and inserted into a eukaryotic expression vector pcDNA3. Again, the placement of HBcAg gene in the resulting plasmid pcDNA-HBc was tested with restriction enzyme digestion. For this purpose, EcoR I and HindUl enzymes which were also utilized in the insertion of the gene into the plasmid in the first place, were used. Afterwards, pcDNA-HBc was transfected into Vero cells. Cells stably transfected with pcDNA-HBc plasmid (Vero-HBc) were seleceted in geneticin containing culture medium. The presence of HBcAg gene in Vero-HBc cells were again confirmed with PCR assay. Additionally, the expression of HBcAg gene by Vero-HBc cells were tested in Western Immunoblotting assay. The result of immunoblotting assay demonstrated that, Vero-HBc cells indeed express a 21 kDa protein reacting with anti-HBcAg antibody. In conclusion, in the present study, it was shown that HBcAg gene of hepatitis B virus, one of the most serious health hazards of modern day world, was cloned in E.coli and expressed in Vero cells. The expressed HBcAg seems to have authentic antigenic properties as determined by Western Immunoblotting assay.