Gene Expression and Activities Analysis of a New Fusion Protein (RGD)3/tTF

Abstract:

To develop a new fusion protein （RGD）3/tTF for the therapy of the selective thrombosis of tumor blood vessels. The fused gene (RGD)3/tTF was reconstructed by PCR, was cloned into vector pET22 b（+），and expressed in E.coli BL21(DE₃）. The fusion protein was purified through Nickel-affinity chromatography column. The tTF activity of the fusion protein was detected by clotting assay and FⅩ activation assay. The specific binding of (RGD)3/tTF to α_ｖβ₃ was analyzed by indirect ELISA. The recombinant plasmid pET22 b（+）/(RGD)3/tTF was obtained and expressed in E. coli BL21（DE₃）. The purified fusion protein could induce blood coagulation, activiate FⅩ. The ability of (RGD)3/tTF binding specifically to α_ｖβ₃was increased by 32%, compared with RGD/tTF. A new fusion protein (RGD)3/tTF was successfully expressed in E.coli BL21（DE₃）. The expressed proteins retained tTF activity and showed a higher binding to α_ｖβ₃ than that of RGD/tTF.