Construction and Identification of Adenoviruses with Ki-67 Core Promoter-regulated E1 A Expression
Objective To construct adenoviral Ad-pki-67-E1 A-GFP containing the promoter of the ki-67gene to achieve replication specifically in ki-67-positive glioma cells.Methods The promoter sequence of the ki-67gene was cloned into the pGL3-basic vector using molecular biology methods.The ki-67 promoter activity was detected by the dual luciferase reporter gene as-say.The ki-67 promoter was constructed into a shuttle plasmid and co-transfected with a helper plasmid in 293 T cells to recombinant adenovirus Ad-pki-67-E1A-GFP.The Ad-pki-67-E1A-GFP was added to glioma cells,and the expression of GFP was ob-served by fluorescence microscopy.Meanwhile,the expression of ki-67gene,the expression of E1A gene,which was essential for adeno-virus replication,and the number of virus copies were detected by real-time fluorescence quantitative polymerase chain reaction(PCR).Results The cloned ki-67sequence was able to activate reporter gene expression in glioma cells;enzymatic digestion and sequencing demonstrated that the ki-67 promoter was constructed into a shuttle plasmid and recombined to obtain the adenovirus Ad-pki-67-E1A-GFP.Ad-pki-67-E1A-GFP could infect glioma cells,and the expression of E1A and the number of virus copies were posi-tively correlated with the expression of ki-67 in the cells.Conclusion The ki-67 promoter-modified adenovirus replicated in glioma cells,providing a basis for further modification for gene therapy of malignant gliomas.
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维普
