Generation of adenovirus-mediated HBsAg gene modified dendritic cells

Objective:To construct recombinant adenovirus vectors containing human HBsAg genes, and infect dendritic cell. Methods: Full length HBsAg cDNAs were subcloned into pIND vector , followed by being cloned into shuttle2 vector. The HBsAg gene fragments resulted from the shuttle2-S digested with PI-Sce and I-Ceu were linked to the linear adeno-X virus DNA. After packaged with HEK293 cells, the adenovirus expression vector was obtained. The plasmid pAdeno-S was identified by endonuclease and PCR.After dendritic cells were infected pAdeno-S, the expressive activity of adenovirus vector was identified by Western blot. And apoptotic analysis was performed in recombinant pAdeno-S infected DCs by Flow cytometric. Results: HBsAg gene in the inserted DNA of adeno-S was confirmed by PCR, and predictive fragments proved by restriction enzyme digestion analysis were exhibited. All the above results indicated that human HBsAg gene had been connected with pAdeno-X vectors correctly. The recombinant adenovirus vector of human HBsAg gene packaged in HEK293 cells, it will be used to introduce the target gene into dendritic cell.Western blot analysis showed that HBV surface antigen gene was expressed in transfected DCs.And adeno-S infection had no appreciable effect on apoptosis of DCs. Conclusion: The recombinant adenovirus vector of human HBsAg gene have been constructed successfully.The established HBsAg -DC vaccine may be a tool of the hepatocellular carcinoma immunotherapy,and it will be foundation of future clinical use of DC vaccine.