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

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pET22b(+)载体,表达于E.coliBL21(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αvβ3的特异结合能力。pET22b(+)/(RGD)3/tTF重组质粒成功获得并表达于E.coliBL21(DE3)。纯化蛋白(RGD)3/tTF能有效诱发血液凝固,活化FⅩ。(RGD)3/tTF与αvβ3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αvβ3的结合能力。

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 （ DEs ）. The fusion protein was purified through Nickel-affinity chromatography column. The tTF activity of the fusion protein was detected by clotting assay and F X activation assay. The specific binding of （RGD）3/tTF to avβ3 was analyzed by indirect ELISA. The recombinant plasmid pET22 b（ ＋ ）/（RGD） 3/tTF was obtained and expressed in E. coil BI21 （DEs）. The purified fusion protein could induce blood coagulation, activiate F X. The ability of （RGD）3/tTF binding specifically to avβ3 was increased by 32%, compared with RGD/tTF. A new fusion protein （RGD）3/tTF was successfully expressed in E. coil B[21 （DEs）. The expressed proteins retained tTF activity and showed a higher binding to avβ3 than that of RGD/tTF.