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Selective Thrombosis in Tumor Vessels Induced by Fusion Proteins tTF-EG3287 Targeting Neuropilin-1

Author: HeJie
Tutor: YanJiangHua
School: Xiamen University
Course: Biochemistry and Molecular Biology
Keywords: tTF Of Neuropilin - 1 Solid tumors Blood clots
CLC: R73-3
Type: Master's thesis
Year: 2009
Downloads: 33
Quote: 0
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Abstract

Selective embolization of tumor blood vessels and induce ischemic necrosis of the tumor tissue is a promising anti-tumor strategy. The oriented vector construction vector-TTF fusion protein will be truncated tissue factor ttf selectively target tumor vascular membrane surface, can induce tumor tissue blood clots blocking tumor of nutrients and oxygen supply, causing the tumor ischemic necrosis , anti-tumor effect. Neuropilin-1 (NRP-1) is a relative molecular mass (Mr) of 120-130 kDa transmembrane glycoprotein, was found to have high expression of the tumor endothelial cells and tumor cells. Of Neuropilin-1 is a non-receptor tyrosine kinase, vascular endothelial growth factor 165 (VE GF165) overexpression of tumor angiogenesis and growth, NRP-1 may become the target of anti-cancer therapy. Haiyan Jia design short peptides EG3287, with the occurrence of Neuropilin-1-specific binding, EG3287 is a fragment of VEGF165, it retains the ability to bind to NRP-1, but does not trigger intracellular signaling pathways, for we use fusion proteins TTF-EG3287 EG3287 as a targeting vector was constructed selectively induce tumor vascular thrombosis. In order to obtain the tTF-EG reorganization of 3287 genes and the active protein product, we used the PCR technique EG3287 with tTF fusion gene was constructed, cloned gene to the original expression vector pET 32b (), IPTG induction of expression by expression condition was optimized to achieve target protein in E. coli BL21 (DE ₃) in the high-level expression, and found the target protein expressed as inclusion bodies, refolding protein was purified by nickel affinity chromatography, dialysis method. Coagulation experiments and F Ⅹ activation experiments confirmed that the fusion protein retained tTF part of the activation of F Ⅹ and lead to blood clotting activity; immunofluorescence assay results showed that ECV304 cell membrane can bring red fluorescent fusion protein TTF-EG3287, equimolar concentrations of TTF protein can not be described EG3287 portion of the fusion protein maintains biological activity, specific binding occurs with the ECV304 cells. The in vivo experiments showed that the fusion protein TTF-EG32 87 can effectively inhibit the growth of murine sarcoma S180, S180 mouse model, TTF-EG3287 inhibition rate reached 64.6%. Histological observation of the fusion protein can be selectively positioned in the tumor tissue and selectively induce tumor vascular thrombosis, leading to tumor cell necrosis, and no toxic side effects on normal tissues. In conclusion, we successfully by genetic engineering techniques to build tTF-EG3287 fusion gene and was highly expressed in prokaryotic expression system. The EG3287 has significant fusion protein TTF-selectively induce tumor activity of blood clots and inhibition of tumor growth, these new drugs for Therapeutic laid the foundation.

Selective Thrombosis in Tumor Vessels Induced by Fusion Proteins tTF-EG3287 Targeting Neuropilin-1

Abstract

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