Construction Of Tumor-Specific Proliferation-Deficient Adenovirus Vector Containing MDA-7 And Its Primary Study On Selective Expression In Tumor Cells

Objective To develop a replication-deficient adnovirus for gene therapy in which the hTERT promoter was introduced to regulate the expression of the target gene MDA-7, and investigated primarily tumor killing of MDA-7 protein.Methods A replication-deficient adnovirus AdTP-mda7 was constructed by gene technology that human telomerase reverse transcriptase promoter and therapic gene were inserted into shuttle plasmid of type 5 adenovirus; simultaneously AdTP-EGFP was constructed as control. The constructed adenovirus was proliferated in HEK 293 cell. Titer was obtained by TCID₅₀ method after purified by caesium chloride density gradient centrifugation. The expression of MDA-7 in tumor cells was detected by western blot and the killing differentiation of two kinds of adenovirus for tumor cells was assessed by violet staining test. Cells were photographed after staining.Results Adenovirus shuttle plasmid pSuTP-mda7 including target gene were successfully constructed and identified by PCR technology. And adenovirus AdTP-mda7 and AdTP-EGFP were obteined successfully. Titer of AdTP-mda7 was 2.69×10⁹pfu/ml and that of AdTP-EGFP was 1.8×10⁸ pfu/ml. Western blot analysed that MDA-7 selectively expressed in various tumor cell lines SGC7901, A549 and so on, inducing tumor cells death while primary lung fibroblast treated in the same condition did not express MDA-7 in detectable levels; violet staining test verified directly that MOI of mass tumor cells infected AdTP- mda7 died was 10, and that infected control adenovirus was 10 000.Conclusion Replication-deficient adenovirus, AdTP-mda7, was successfully constructed, simultaneously verified that the expression of MDA-7 in tumor cells induced tumor cell death, which was great significant for tumor-targeted gene therapy.