Construction Of Double-regulated Chimeric Oncolytic Adenovirus With TNF-? Gene And Killing Effect On Gastric Cancer Cells

AIM To develop an double-regulated Ad5/35 chimeric oncolytic adenovirus with TNF-a gene and observe its cytotoxicity in gastric cancer cells.METHODS the human TNF-a gene was obtained with reverse transcription polymerase chain reaction(RT—PCR)from the PCD DNA-TNF-? plasmid,the TNF-? gene was recombined to construct the transfer plasmid PPE3-F35-TNF-? by molecular clone technique,the transfer plasmid PPE3-F35-TNF-? and the plasmid SG502-EGFP were contransfected to 293T cells mediated by lipofectamine 2000 to produce oncolytic adenovirus particles.The recombinant adenoviruses were veriffied by PCR,purified by cesium chloride density purification and propaganted in 293 cells.Virus titer was measured by TCID50 method.The killing effect was observed by MTT.The expression of TNF-a was observed by Elisa.RESULTS Restriction endonuclease and PCR analyses confirmed that the TNF-a gene was successfully inserted into the double-regulated Ad5/35 chimeric oncolytic adenovirus vector.The titer of the human TNF-a gene recombinant adenovirus was 2×1011pfu/ml,Obvious apoptosis in cultured cancer cells was induced by PPE3-F35-TNF-a at MOI of 1,and there was no significant effect on MRC-5 even at MOI of 100.The expression of TNF-a by Elisa show that the PPE3-F35-TNF-? group(1474.86±71.33ng/ml),the SG502-EGFP group(14.06±0.92 ng/ml)and the blank control group(15.84±2.57ng/ml)have significant difference between the three groups(P<0.001).CONCLUTION PPE3-F35-TNF-a selectively replicates in carcinoma cells and specifically kills the tumor cells.This will facilitate the following exploratory development of oncolytic adenovirus carrying TNF-a gene based gastric carcinoma.And the PPE3-F35-TNF-a holds great promising in cancer therapy.