Construction of Eukaryotic Expression Plasmids Containing A 1.3-fold Overlength Genome HBV and Its Expression in Bewo Cells in Vitro

Objective To construct a recombinant eukaryotic expression vector pCDNA3.1(+)-HBV1.3, and determine the expression of the recombinant protein in Bewo cells. Methods The 1.3-fold overlength genome HBV DNA sequence was constructed from a recombinant plasmid pMD18T-HBV HBV DNA sequence and cloned into the eukaryotic expression plasmids pCDNA3.1 (+). The recombinant vector was confirmed by restriction enzyme digestion, PCR and sequencing. The plasmid was then transfected into Bewo cells.The level of extracellular and intracellular HBsAg,HBeAg expression and HBV DNA level was detected by Western blotting,Microparticle Enzyme Immunoassay (MEIA) and fluorescence quantitative PCR, respectively. Results The results of the restriction enzyme digestion, PCR and sequencing confirmed that the vector was constructed successfully.It can express and secret HBsAg and HBeAg protein by the Bewo cells.A high level HBV DNA was found in the supernatant.Conclusion The construction of this expression vector will provide the foundation for future research on the prevention of intrauterine HBV infection.