Construction of secretory eukaryotic expression vector of NT4-APOPTIN-HA2-TAT and its effects on tumor cells

C288 Objective :To enhance apoptin’s antitumor therapeutic effect , the Apoptin gene was fused with NT4 and HA2-TAT to construct secretory eukaryotic expression vector,and explore its antitumor effect. Methods : The Apoptin gene were obtained by T-vector method,and then Apoptin and HA2-TAT gene were inserted in pUC19/NT4 vector. The fusion gene of NT4-Apoptin-HA2-TAT was sub-cloned into the shuttle plasmid of adeno-associated virus; the products were co-transferred into HEK-293 cell line with helper plasmid pAAV/Ad and adeno plasmid pFG140.The recombinant adeno-associated virus was produced by homologous recombination of above 3 plasmids in HEK-293 cells and its titer was measured by quantitative dot blot hybridization. The effects of AAV-NT4-Apoptin -HA2-TAT on human HepG2 cell line and mouse fibroblast cell NIH3T3 were measured by MTT assay and Flow cytometry assay. Results: The vector was confirmed by restriction enzyme digestion and DNA sequencing. High titer of recombinant adeno-associated virus was obtained by homologous recombination in HEK-293 cells(3.14×1011pfu/ml). It could induce apoptosis after HepG2 cells were transfected in 48h later, but not in normal NIH3T3 cells, which significantly decreased the survival rate of HepG2 cells. Conclusion: The secretory recombinant adeno-associated virus vector encoding gene NT4-Apoptin-HA2-TAT has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques,which can be secreted and induces apoptosis of tumor cells specificity, laying a foundation for further research of gene therapy of cancer.