A recombined fusion protein SP5.2/tTF induce thrombosis in tumor blood vessel.

Tumor vascular targeting is one of the most promising strategies in tumor therapy. Here we used E.coli to express a recombinant SP5.2/tTF fusion protein, which, as a tumor vascular targeting agent, consists of SP5.2 (a peptide selectively binding and targeting VEGFR-1 on tumor endothelial cells) and truncated tissue factor (tTF)and aimed to explore its anti-tumor activities.The SP5.2/tTF expression construct was synthesized by polymerase chain reaction (PCR) and recombined into plasmid pET22b(+). The fusion gene was verified by restriction mapping and sequencing. SP5.2/tTF was expressed in E. coli and then purified on a nickel-affinity chromatography column. The purified product was detected by SDS-PAGE. The pro-coagulant activity and binding of SP5.2/tTF to human umbilical vein endothelial cells (HUVECs) were monitored by FX activation analysis and fluorescent scanning confocal microscopy, respectively. The effect of SP5.2/tTF on tumor growth was analyzed in BALB/c mice bearing sarcoma 180 (S180) tumor. The tissue localization of SP5.2/tTF and its effect on tumor vessel thrombosis were observed by in vivo fluorescence imaging and histological studies, respectively. The fusion gene was successfully cloned into pET22b(+). SP5.2/tTF was abundantly expressed in bacterial cells and efficiently purified by nickel-affinity chromatography. Functional studies showed that the protein retained both the coagulation activity of tTF and the binding capacity of SP5.2 to HUVECs. In tumor xenograft studies, SP5.2/tTF selectively targeted the tumor, induced thrombosis, and led to retardation and even regression of tumor growth (growth inhibition ratio = 70%, P< 0.05). The recombinant fusion protein SP5.2/tTF inhibited tumor growth by selectively inducing thrombosis in tumor blood vessels.