Construction and identification of conditionally replicating adenovirus vector Ad-delE1b55kD-shRNA/Survivin-EGFP / 第三军医大学学报

ABSTRACT

Objective To construct the conditionally replicating adenovirus vector Ad-delE1b55kD-shRNA/Survivin-EGFP that can transfects into HT-29 effectually and selectively and contains shRNA targeting to human Survivin gene.Methods EGFP geoe and shRNA gene were inserted into pAd-delE1b55kD,then pAd-delE1b55kD-shRNA/Survivin-EGFP was obtained.The plasmid and pBHGE3 were cotransfected into HEK-293 cell to obtain the conditionally replicating adenovirus vector Ad-delE1b55kD-shRNA/Survivin-EGFP.Then DNA of the adenovirus vector was extracted and identified.The transfection efficiency of the vector to HT-29 and LO2 was detected.The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot.Results EGFP gene and shRNA gene targeting to human Survivin mRNA were inserted into conditionally replicating adenovirus vector successfully,proven by sequencing.The transfection efficiency to HT-29 of Ad-delE1b55kD-shRNA/Survivin-EGFP was significantly higher than replication defective adenovirus and liposome vector(P