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

S. LV, M. YE, X. WANG, Z. LI, X. CHEN, X. DOU, Y. DAI, F. ZENG, L. LUO, C. WANG, K. LI, X. LUO, J. YAN, X. LI

Abstract:

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%, PThe recombinant fusion protein SP5.2/tTF inhibited tumor growth by selectively inducing thrombosis in tumor blood vessels.

Issue: 4/2015

Volume: 2015

Pages: 531 — 540

DOI: 10.4149/neo_2015_064

Pubmed

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