一种新型融合蛋白（RGD）3/tTF的基因表达及活性分析

[Gene expression and activities analysis of a new fusion protein (RGD) 3/tTF].

作者信息

Yan Jiang-Hua, Yang Gui-Wang, Wang Jie-Ping, Wu Na, Zhuang Guo-Hong

机构信息

Cancer Research Center of Medical School, Xiamen University, Xiamen 361005, China.

出版信息

Sheng Wu Gong Cheng Xue Bao. 2007 May;23(3):409-12. doi: 10.1016/s1872-2075(07)60032-6.

DOI:10.1016/s1872-2075(07)60032-6

PMID:17577984

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 F X activation assay. The specific binding of (RGD) 3/tTF to alphavbeta3 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 F X. The ability of (RGD) 3/tTF binding specifically to alphavbeta3 was 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 alphavbeta3 than that of RGD/tTF.

摘要

开发一种用于治疗肿瘤血管选择性血栓形成的新型融合蛋白（RGD）3/tTF。通过PCR重建融合基因（RGD）3/tTF，将其克隆到载体pET22 b(+)中，并在大肠杆菌BL21（DE3）中表达。通过镍亲和层析柱纯化融合蛋白。通过凝血试验和F X激活试验检测融合蛋白的tTF活性。通过间接ELISA分析（RGD）3/tTF与αvβ3的特异性结合。获得重组质粒pET22 b(+)/(RGD)3/tTF并在大肠杆菌BL21（DE3）中表达。纯化的融合蛋白可诱导血液凝固，激活F X。与RGD/tTF相比，（RGD）3/tTF特异性结合αvβ3的能力提高了32%。新型融合蛋白（RGD）3/tTF在大肠杆菌BL21（DE3）中成功表达。表达的蛋白保留了tTF活性，并且与αvβ3的结合能力比RGD/tTF更高。