Recombinant immunotoxin VEGFI65-PE38 in antiangiogenic therapy for cancer

Abstract

Objective To construct a new recombinant immunotoxin expression vector by fusing human VEGFI65 and a truncated pseudomonas exotoxin A ramification (PE38) gene, and explore the in-fluence of the VEGFI65-PE38 fusion protein on human umbilical vein endothehal cells (HUVECs). Methods VEGFI65 was cloned by polymerase chain reaction (PCR). PE38 gene was gained from a vec- tor plasmid pRB391 by restriction endonuelease digestion, and then inserted to the eukaryotic expression vector plRES2-EGFP. The vector was transfected into 293 cells. RT-PCR and ELISA were used to confirm the expression of the fusion gene in the 293 cells. The selectively killing activities of the immunotoxin in culture supernatant were detected in vitro. Results The fusion gene eukaryotie expression plasmid was constructed successfully. The fusion gene could be expressed in the 293 cells. The VEGF levels of(269.0 ± 23.6 ),(306.0 ± 29.3)and(1390.0 ± 136.6 )ng/ L were secreted into the culture medium by no transfected cells, plRES2-EGFP-transfected cells and VEGFI65PE38 transfeeted cells respectively. VEGF165-PE38-containing supematant was specific to VEGFR-positive HUVECs, and the apoptosis rate was 8. 34% , 7. 69%, and 39. 88% in no transfected group, plRES2-EGFP-transfected group and VEGFI65PE38 transfeeted group respectively. Conclusion The results provide the basis for research of the targeted cytotoxie activity to tumor vascular endothelial cells, and may have some potential values in clinical application. Key words: VEGF; Fusion gene; Umbilical vein