Study On Effect Of Targeting Adenovirus Vector Carrying Apoptin Gene On Colon Carcinoma Cell

Objective:To construct recombinant adenovirus vector of CEA promotor controlling the VP₃ gene's expression,and to investigate the targetting effects of the recombinant adenvirus vector on the human colon carcinoma cell.Methods:The pCEA and VP3-PolyA genes were obtained through PCR amplification,and connected to pCEA-VP3-PolyA(to call it CVP for short) by overlapping PCR.The CVP gene was cloned into adenovirus shuttle plasmid pAdtrack,then the resultant plasmid pAdtrack-CVP was cotransformed into E.coli BJ5183 bacterium with adenovirus backbone plasmid pAdEasy-1.The recombinant adenovirus plasmid pAd-CVP was digested with pacI and transfected into HEK293 cells to package recombinant adenovirus particles.The recombinant adenovirus(Ad-CVP) was amplified,purified and the titer was determined.The human colon carcinoma cells LS 174T, human hepatoma cells Hep G2 and HEK293 cells were infected by the recombinant adenovirus, and the human colon carcinoma cells LS 174T were infected by PBS and controlling virus for control.The VP3 gene expression both from the mRNA level and the protein level was detected by RT-PCR and WESTERN BLOT.Qualitative detection of apoptosis was made by Hoechst33258 staining and Quantitative detection of apoptosis was made by flow cytometer (FCM).The difference of apoptosis in different kind of cells was compared.Results:Restrictive enzyme digestion and sequencing results showed that the pCEA and VP3-PolyA genes were correctly obtained,and the two gene was correctly spliced into CVP. Resultant plasmid pAdtrack-CVP was also correctly constructed.Recombinant plasmid pAd-CVP was constructed by homologous recombination in E.coli BJ5183,and recombinant adenovirus was obtained by transfecting HEK293 cells.The titer of adenovirus Ad-CVP was 1.5×10¹¹pfu/ml.In 24h after infection,VP3-mRNA and VP3-protein were detected in the human colon carcinoma cells,while they weren't detected in the PBS group,the control virus group and the other two kind of cells.The apoptotic morphologic change was demonstrated in human colon carcinoma cells by stain with Hoechst33258,while it wasn't demonstrated in the PBS group,the control virus group and the other two kind of cells.The apoptotic rate of the human colon carcinoma cells LS 174T,human hepatoma cells Hep G2 and HEK293 cells was,in turn, 36.9%±2.1%,5.3%±1.3%and 4.9%±0.9%.The apoptotic rate of the human colon carcinoma cells LS 174T was much higher than the other two kind of cells(P＜0.05).Conclusion:It is concluded that the recombinant adenovirus Ad-CVP specifically induces human colon carcinoma cells(CEA+) apoptosis.