Construction and identification of a double-regulated replicative adenovirus AdTPHre-hEndo / 中华肝胆外科杂志

ABSTRACT

Objective To develop a double-regulated replicative adenovirus carrying the Human endostatin gene(hEndo). Methods The plasmid pTPHre-hEndo was constructed by gene engineering technique, carrying human endostatin gene, in which El A gene and E1B gene were driven by human telomerase reverse transcriptase (hTERT) promoter and hypoxia response element (HRE) promoter,respectively. The pTPHre-hEndo was co-transfected with pBHGE3 in 293 cells to generate recombinant adenovirus AdTPHre-hEndo. Virus titer was measured by the TCID50 method. Virus replication assay was performed to evaluate the selective replication ability of AdTPHre-hEndo. The transgene expression of endostatin was detected by ELISA assay. Results A novel gene-viral therapeutic system AdTPHre-hEndo was constructed by gene engineering technique and its titer was 3. 25 X 1010 pfu/ml.Proliferative test revealed that AdTPHre-hEndo could proliferate selectively in telomerase positive tumors. Furthermore, in comparison with non-replicative adenovirus Ad-hEndo, the transgene expression of endostatin mediated with AdTPHre-hEndo was significantly increased (P < 0. 01).Conclusion The novel gene-viral therapeutic system AdTPHre-hEndo has the capacity to replicate in pancreatic cancer cells and expresses the endostatin efficiently, and may provide a new strategy for pancreatic cancer gene therapy.