Construction of recombinant adenovirus vector containing human survivin gene and expression in dentritic cells / 第三军医大学学报

ABSTRACT

Objective To construct the recombinant adenovirus vector containing human survivin, and transfect it into dendritic cells. Methods Full length survivin cDNA was obtained from recombinant plasmid pCITE-survivin by PCR. The PCR product was double-digested with restriction endonucleases KpnⅠ and XholⅠ, and inserted orientationally into pAdTrack-CMV. The plasmid of pAdTrack-survivin was lined with PmeⅠ, and the fragment containing survivin was reclaimed and transfected into E. coli. BJ5183. After having been screened, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by the green fluorescence protein (GFP) expression and by PCR method. The virus was transfected into dentritic cells, and the expression of survivin was proved by the GFP expression and Western blot analysis. Results The recombined adenovirus-survivin was constructed successfully and the titer was about 1.65?10 8 pfu/ml. A band was observed by Western boltting and its relative molecular mass was about 16.5?10 3. Conclusion A recombinant adenovirus vector containing human survivin was constructed successfully, and the survivin protein was expressed by dendritic cells.