Construction of replication-deficient recombinant adenoviral vector carrying HBcAg gene and its expression in vitro

[Objective] To construct recombinant adenoviral vector carrying HBcAg gene by homologous recombination in bacteria and to detect its expression in vitro.[Methods] HBV C genes were amplified by PCR and were cloned to adenoviral shuttle plasmid pAdTrack-CMV.Then the resultant pAdTrack-CMV-HBc was cotransfected into BJ5183 bacteria with the plasmid pAdeasy-1.The adenoviral plasmid carrying HBV C gene(pAd-HBc) was generated with homologous recombination in bacteria and the adenoviruses were produced in 293 cells.Several kinds of mammal cells(293 cells,Vero cells,HepG2 and MSCs) were infected with adenoviruses and the expression of HBcAg was detected by RT-PCR and FACS in vitro.[Results] The adenoviral plasmids pAd-HBc were obtained by selection for kanamycin resistance and confirmed by restriction endonuclease PacⅠanalyses.The recombinant adenoviruses Ad-HBc were packaged successfully in 293 cells.The titer of Ad-HBc was up to 5×109 pfu/mL after the second generation of proliferation in 293 cells.HBcAg were expressed efficiently in mammal cells after infection.[Conclusion] The recombinant adenoviruses expressing HBcAg were constructed successfully and can be used in further study of gene therapy for HBV.