Construction of A Recombined Adenovirus Vector Carrying HBV-S Gene and Its Expression of HBsAg in Dendritic Cell

Objective To construct the recombined adenovirus vector carrying HBV-S gene,which can express HBsAg efficiently,and demonstrate that it can be expressed in dendritic cells for the use of experimental study in gene therapy.Methods By using AdEasy~(TM) XL adenovirus vector system,a recombined shuttle vector was constructed through the clone of HBV-S gene into P-shuttle-CMV,then the vector was linearized using PmeⅠand transformed into BJ5183-AD-1 cell to generate recombined adenovirus(Ad-S) plasmid through electroporation.Subsequently,the plasmid was linearized with PacⅠand transfected into AD293 cell to pack into recombined adenovirus(Ad-S).Ad-GFP or Ad-S at a MOI of 100 transfected dendritic cells,which derived from murine bone marrow cells and proliferated by the induction of cytokines,such as GM-CSF、IL-4 and TNF-α.Lastly,the expression of GFP by Ad-GFP-transduced DCs was detected by flow cytometry,and the expression of HBsAg by Ad-S-transduced DCs was determined by Western blotting,RIA and ELISA.Results PCR,DNA sequence and restriction endonuclease digestion analysis indicated that the recombined adenovirus vector carrying HBV-S gene had been constructed successfully.The virus particle titre of Ad-S was 10~8 /ml.The expression rate of GFP by Ad-GFP-transduced DC was(92.93±2.35)% detected by flow cytometry and fluorescence density reached to 236.67±45.72.Western blotting,RIA and ELISA all had detected HBsAg expressed by Ad-S-transduced DCs.Conclusions Success of the Ad-S construction and expression of HBsAg by Ad-S-transduced DCs lays the foundation for future investigation of the feasibility of Ad-S-transduced DCs as an HBV immunotherapeutic vaccine.