中文摘要：

目的：用大肠杆菌表达骨桥蛋白RGD黏附序列6拷贝短肽，经分离纯化后检测其生物学活性。方法：运用基因重组技术，将骨桥蛋白RGD黏附序列的核酸片段首尾相连，与携带GST编码序列的原核表达载体连接构建融合蛋白表达质粒pGEX，3X—RGD。将重纽质粒转化宿主菌后，对诱导融合蛋白表达的条件进行优化。表达产物GST—RGD经谷胱甘肽一亲和层析纯化后，分别检测其对骨桥蛋白诱导的血管平滑肌细胞黏附和迁移的影响。结果：所构建的含有6个拷贝短肽的GST-RGD融合蛋白可在大肠杆菌中以包含体的形式进行袁达。用十二烷基肌氨酸钠变性溶解包含体及透析复性后，经亲和层析可得到高纯度的GST—RGD（6）融合蛋白。GST—RGI）（6）融合蛋白能特异性的押制骨桥蛋白诱导的血管平滑肌细胞的黏附和迁移。结论：骨桥蛋白RGD黏附序列6拷贝短肽可在大肠杆菌中高效表达，纯化的GST—RGD融合蛋白具有抑制血管平滑肌细胞黏附和迁移的活性。

英文摘要：

Aim： The six copies of RGD sequences present in osteopontin were expressed in the E. coil, and the biological activity of the purified products was studied. Methods： cDNA fragments containing six copies of RGD sequences were subcloned into prokaryotic ex pression vector pGEX-3X including GST coding sequence to construct pGEX-3X-RGD plasmids. E. coil DH5α transformed by pGEX- 3X-RGD（6） plasmid was induced by different IPTG concentrations for different times to identify the optimal induction condition. Induced GST-RGD fusion protein was purified via GST-Sepharose 4B affinity resin. Results： GST-RGD fusion proteins containing six copies of RGI） sequences could be successfully induced and were mainly located in inclusion bodies. After being denatured and dia lyzed, renatured fusion proteins were purified via GST-Sepharose 4B affinity resin. GST-RGD（6） fusion protein could specifically inhibit adhesion and migration of VSMC stimulated by osteopontin, which could be considered as a basis on producing small peptides containing RGD sequences for inhibition of VSMC adhesion and migration. Conclusion： The six copies of osteopontin RGD peptide were successfully expressed in E. coli DH5α. The purified GST-RGD fusion protein could inhibit the adhesion and migration of VSMC stimulated by osteopontin.