Construction and identification of recombinant adeno-associated virus vector mediating the expression of the derepressing p73 peptide-p53(N37)

Objective To construct a recombinant adeno-associated virus vector mediating the expression of the derepressing p73 peptide-p53(N37) so as to lay a foundation for further research on gene therapy of malignant tumors. Methods The p53(N37) gene was obtained by self-complementary primer PCR and T-vector cloning techniques;then the p53(N37) gene and HA2-TAT segment were cloned into pUC19/NT4 vector after digested with restriction enzyme.The fusion gene of NT4-p53(N37)-HA2-TAT was subcloned into the shuttle plasmid of adeno-associated pSSHG-CMV,and recombinant plasmid pSSHG-CMV/NT4-p53(N37)-HA2-TAT was constructed and identified by enzyme cutting analysis.The rAAV/NT4-p53(N37)-HA2-TAT was produced by using calcium phosphate coprecipitation method and HEK 293 cell line was co-transfected by pSSHG-CMV/NT4-p53(N37)-HA2-TAT,aid plasmid pAAV-Ad and adenovirus genome plasmid pFG140.The virus titer was determined by dot-blot hybridization.The effect of rAAV/NT4-p53(N37)-HA2-TAT on HepG2 cell line was measured by a methyl thiazolyl tetrazolium(MTT) assay and flow cytometry. Results The p53(N37) gene was confirmed by restriction enzyme digestion and DNA sequencing.High titer of recombinant adeno-associated virus was obtained by homologous recombination in HEK293 cells(2×1013pfu/L).The rAAV/NT4-p53(N37)-HA2-TAT had a notable lethal effect on HepG2 cells and this effect was realized by inducing the apoptosis of HepG2 cells. Conclusion The recombinant adeno-associated virus vector mediating the expression of the derepressing p73 peptide-p53(N37) has been successfully constructed by molecular cloning and in vitro recombination techniques in this experiment,which lays a foundation for further research on gene therapy of malignant tumors.