Construction Of Tumor-specific Proliferation Adenoviral Vecto

A number of features unique to adenoviruses contribute to their utility as gene-transfer vectors,including tranducing both dividing and non-dividing tumor cells,not integrate into the cell genome,no insert mutagenicity,high safety,high efficiency,low toxicity,high-capacity,high specificity,and low degradation in cells.We used human telomerase reverse transcriptase(hTERT)promoter and hypoxia response(HRE)promoter to regulate the expression of E1A and E1B,which are associated with the proliferation of adenovirus the genes,of adenovirus respectively.Therefor,the replication of adenovirus is confined to the telomerase-positive and hypoxia tumor cells.Specific experimental methods are:used two overlapping PCR site-directed mutagenesis techniques to delete the wild-type adenovirus E1A and E1B endogenous promoter,then introduced two restriction sites and used the two sites to insert human genome telomerase reverse transcriptase promoter hTERT and hypoxia response promoter HRE up-stream of E1A and E1B genes respectively.In order to facilitate follow-uptesting,we inserted green fluorescence protein reporter gene between the HRE promoter and the E1A protein gene.Finally,we successfully reconstructed a new shuttle plasmid pXCl-hTERT-GFP-HRE.Then Next,we cotransfected the shuttle plasmid pXCl-hTERT-GFP-HRE with the backbone plasmid pBHGE3i containing wild-type adenovirus Ad5 vector lower right arm into 293,so that the two plasmids can homologously recombine in the cell.About 9-10 days after transfection,viral plaques appeared,then collected these cells,repeatedly freezing and thawing the virus.Collected the virus and named it as KGHV200.Then we determined the titer by TCID50 method.Real-time quantitative PCR results showed that,when KGHV200 infecting human hepatoma cell lines HepG2,a higher expression of E1A and E1B genes were found,suggesting that the tumor-specific expression of E1A and E1B gene can be successfully regulated by the two tumor-specific promoter we constructed.Virus proliferation experiments suggested,by infecting the human embryonic lung diplod fibroblast KMB17 and several strains of telomerase-positive tumor cells(as MCF-7,MDA-MB-435,XWLC-05,HepG2 and HCT116)respectively with KGHV200 carring the GFP reporter gene(MOI=0.1)and observing the GFP expression in the third day,the seventh day,the ninth day and the 12th day under the fluorescent microscopy respectively.The results of fluorescence in human embryonic lung diplod fibroblast dispersed clusters of a single;in tumor cells,its present a single expression and gradually become widely dispersed,then concentration,presence of a fusion.In 12th day after the fluorescence gradually faded,floating dead cells into groups.Virus proliferation experiments:infected human embryonic lung diplod fibroblast KMB17 and different tumor cell lines:including breast cancer cell line MDA-MB-435,human hepatoma cells HepG2,human colon cancer HCT-116,Sherwin and lung XWLC-05 human gastric cancer cell lines SGC9701 with KGV200 of the same titer(MOI=5).After infection of 24h,48h,and 100 hours,collected the virus and determined its titer by TCID50.The results showed that KGHV200 can massively replicate in different tumor cell lines,while not in normal cells.In summary,our laboratory successfully constructed an oncolytic virus which regulated by two tumor-specific promoter.In vitro experiments showed that the recombinant adenovirus can highly express E1A and E1B gene in tumor cells.And the recombinant adenovirus efficiently replicated and proliferation in different tumor-specific cells.It provides an ideal transport carrier for gene therapy.