Construction of a pcDNA-HBeAg expression vector to express HBeAg in CHO cells

Objective To clone the hepatitis B e antigen (HBeAg) gene from the hepatitis B virus (HBV) genome into a eukaryotic expression vector so that the HBeAg gene may be efficiently expressed in the Chinese hamster ovary (CHO) cell line for in vitro analyses.Methods The HBeAg gene was PCR amplified from HBV DNA extracted from the serum of an HBeAg-positive patient and using primers based on the HBV strain FMU013.The purified amplicon was inserted into the pcDNA3.1 (+) eukaryotic expression vector.The resultant recombinant plasmid, pcDNA-HBeAg, was verified by restriction enzyme digestion and PCR sequencing, and then transformed into cultured CHO cells.Expression of the HBeAg protein was confirmed by PCR, enzyme-immunodotting assay, Western blotting, and immunocytochemistry.Results The HBV HBeAg gene was successfully amplified and cloned to generate the pcDNA-HBeAg eukaryotic expression vector.Nucleotide sequencing analysis of the full-length HBeAg gene clone revealed 12 base substitution mutations (C1819G, A2007T, C2046T, C2061G, G2106A, C2109A, C2146T, T2172C, C2203T, A2235G, G2253A, and C2298T) and 1 deletion mutation (2346 del T) .Amino acid sequence analysis of the mature HBeAg protein revealed only one mutation, which was a valine (V) to phenylalanine (F) substitution at site 149 (V149F) .The V149F mutation formed a peptide (RLESRGPVZTR) that was fused to the carboxyl terminus of the HBeAg protein.The fusion HBeAg protein was expressed in CHO cells, and was detected by PCR, enzyme-immunodotting, Western blotting, and immunocytochemistry tests.Conclusion The recombinant pcDNA-HBeAg vector successfully expresses the fusion HBeAg protein in CHO cells.This newly developed in vitro system is a potentially useful and convenient tool for further study of HBeAg.