Construction of HBsAg gene recombinant adenoviruses and packing in 293 cells / 中国免疫学杂志

ABSTRACT

Objective:After construction and identification of HBsAg gene recombinant backbone adenoviral vector,it is to prouduct HBsAg gene recombinant adenoviruses by packing PAd-Easy-1-HBs in 293 cells.Methods:The gene of interest was amplified from plasmid pEcob-6 PCR, the gene of interest which contained HBsAg gene was cloned into on adenoviral shuttle vector pAd-track-cmv. The pAd-track-cmv-HBs was linearized by digesting with restriction endonuclease Pme-1, and subsequently cotransformed into E.coli BJ-5183 cells with an adenoviral backbone vector pAd-Easy-1, Homologous recombinants were performed in bacterial cells. Finally, the linearized backbone adenoviral vector was transfected into adenoviruses packing cells lines,e.g. 293 cell by lipofectamine transfection. Transfections and viral productions can be minotored by green fluorescent protein(GFP). The expression of HBsAg in supermatant was investigated by ELISA. It was a certain HBsAg vaccine to amplify recombinant adenoviruses by repeating the infevtion cell to collect the viral supermatant.Results:GFP expression was visible by fluorescence microscopy after transfection. Adenoviral titer was monitored by GFP expression. GFP expression was visible after repeating the infection cell using the viral supernatant in more than 90 percent of the cells.The HBsAg also expressed in supermatant.Conclusion:HBsAg gene recombinant adenoviral backbone vector has been constructed successfully. HBsAg gene recombinant adenoviruses have been producted by packing in 293 cells. The study provides the possibility of further researches on the development of new anti-HBV vaccines.