一种新型融合蛋白(RGD)3/tTF的基因表达与活性分析

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

摘要:

为了发展一种新型的融合蛋白(RGD)3/tTF用于肿瘤血管的选择性栓塞治疗，利用PCR技术重组(RGD)3/tTF融合基因，克隆于pET22 b（+）载体，表达于E.coli BL21(DE₃）。用镍柱纯化融合蛋白。凝血实验与FⅩ活化实验检测融合蛋白tTF组分的活性。间接ELISA分析(RGD)3/tTF与 α_ｖβ₃的特异结合能力。pET22 b（+）/(RGD)3/tTF重组质粒成功获得并表达于E. coli BL21（DE₃）。纯化蛋白(RGD)3/tTF能有效诱发血液凝固，活化FⅩ。(RGD)3/tTF与α_ｖβ₃的特异结合能力比RGD/tTF提高了32%。 新型融合蛋白(RGD)3/tTF已在E. coli系统成功表达，表达蛋白保持tTF的活性并显示比RGD/tTF更高的与α_ｖβ₃的结合能力。

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.