Selective Thrombosis In Tumor Blood Vessels Induced By Fusion Proteins (CREKA)₃-tTF

It is a promising anti-tumor strategy to selectively shut down the blood vessels of tumors.This strategy use truncated Tissue Factor(tTF) as the effector,which combined with tumor vessels targeting carrier to selectively induce thrombosis in tumor vasculature and cut off the nutrition or oxygen supplies of tumor,subsequently result in necrosis of the tumor and obtained anti-tumor effects.The specific ligands are needed to deliver tTF to tumor vessels in the strategy. The CREKA peptide can target of resident clotted plasma proteins in the tumor vasculature.So the CREKA is a well deliver for tTF.It is expected that CREKA/tTF fusion protein has anti-tumor effects by selectively inducing thrombosis in tumor vasculature.Firstly,The CREKA/tTF fusion proteins were designed and analysed by molecular simulations using bioinformatics applications.The optimal model was chose to build Secondly,The CREKA/tTF fusion gene was reconstructed by PCR technology and cloned into pET22b(+).Then the CREKA/tTF fusion protein was expressed in E.coil BL21 and purified by His SpinTrap columns.After purify,the fusion protein was refold by subsequent dialysis.Thirdly,the activities of the fusion proteins were measured by coagulation timing,FX activation,and quantitative fluorescence test in vitro.Fourthly,the distribution of the RBITC-labelled proteins in mice bearing a grafted S180 tumor was examined by whole-animal imaging.And the localization of the RBITC-labelled protein in tissues was observed by laser scanning confocal microscope.Finally,the effect of the protein to selectively induce thrombosis in tumor vasculature was examined by histologic analysis.The(CREKA)₃-G₄S -tTF model is chose on the result of molecular simulations. Both the protein fragments of CREKA and tTF in the chose model are analysed to be effective of their function by molecular simulations.Then we successfully build the gene sequence in pET22b(+) and express the protein in the E.coil BL21.The coagulation of the fusion protein show in the coagulation test and FX activation test. And the capability of the fusion protein effectively binding to clotted plasma proteins is identified in quantitative fluorescence test.The whole-animal imaging shows the fusion protein is only in the tumor and slices fluorescent indicats the fusion protein is in tumor vessles.I n histochemical analysis thrombosis of tumor blood vessels in (CREKA)₃-tTF treated mice is observed,but not in other normal tissues.A significant reduction in tumor growth rate is observed in(CREKA)₃-tTF treated group,compared with control.In conclusion,our results demonstrate that the(CREKA)₃-tTF is the protein that can target to tumor and cause selectively vascular occlusion in the tumor vessels to gain antitumor effects.