关键词: RGD, tTF, 靶向血栓蛋白, 整合素αv β3

Abstract: BACKGROUND AND AIM: To study the ability of(RGD)3 -tTF thrombosis targeted protein binding to tumor vascular markers such as integrin αv β3, and to investigate the relationship between the targeting ability to integrin αv β3 and the chemical structure of RGD polypeptide of the fusion protein. MATERIALS AND METHODS: The (RGD)3 -tTF fusion gene was synthesized by the truncated tissue factor (tTF) and three RGD polypeptides in series and expressed in Escherichia coli(E.coli)BL21 (DE3). The fusion protein was then purified by Nickel affinity chromatography column. The activity of the tTF fusion protein was evaluated by clotting assay. The binding ability to intergin αv β3 specially was analyzed by indirect enzyme linked immunosorbent assay (ELISA), and compared with the activity of RGD-tTF fusion protein. RESULTS: The coagulation activity of (RGD)3 -tTF and RGD-tTF purified fusion protein was alike (F=0.019，P>0.05). However, the ability of the (RGD)3 -tTF fusion protein binding to intergrin αv β3 specifically was elevated significantly (F=140.17，P<0.01). When the concentration was 0.24 μmol/L, the OD405nm of the (RGD)3 -tTF fusion protein was 1.3 times more than that of RGD-tTF fusion protein(1.25/0.95). CONCLUSION: Because the (RGD)3 -tTF fusion protein has two disufides and three RGD polypeptides, the coagulation activity of tissue factor was retained and the ability binding to integrin αv β3 was increased significantly.It may provide a foundation for the further study of thrombosis- targeted therapy in animal tumor models.

Key words: RGD, tTF, thrombosis-targeting protein, integrins αv β3