人vW因子A3区基因在大肠杆菌中表达及其生物学活性的研究

Expression of von Willebrand Factor-A3 Domain in E coli and Its Biological Function

血管性血友病因子 (vWF)是介导血小板粘附到细胞外基质的桥梁 ,在血栓形成过程中发挥重要作用 ,可通过阻断vWF与细胞外基质的结合而阻断血小板的粘附。本研究目的是研究一种抗血栓形成的新疗法。应用RT PCR方法从人脐带内皮细胞中克隆vWFA3区基因并在大肠杆菌中表达 ;应用胶原结合试验及竞争抑制实验分析rvWF A3蛋白的生物学活性。结果表明 :表达的重组蛋白量占菌体总蛋白的 4 6 % ,包涵体经过变性、纯化和复性 ,获得重组蛋白 (rvWF A3) ;rvWF A3具有很好的胶原结合活性 ,且能竞争性抑制野生型vWF与胶原结合。结论 :大肠杆菌中高效表达的rvWF A3具有良好的生物学功能 ,可作为阻断剂用于干预vWF介导的血小板黏附过程 。

The interaction among collagen, von Willebrand factor(vWF) and glycoprotein Ib axis is the first step in hemostasis and thrombosis, especially under high shear condition. To develop a new remedy of anti thrombosis, mRNA from endothelial cells was extracted, and reverse transcription PCR was adopted to amplify DNA of interest. After sequencing, recombinant expression vector was constructed. The amplified DNA fragment of vWF domain A3 was inserted into expression vector with 6×his taq, pET20b(+), the recombinant was transformed into E coli (strain DE3) and induced by IPTG. Recombinant vWF A3 was designated as a recombinant fragment comprising residues 918 1114 of mature vWF subunit. It was purified through Ni NTA resin column and refolded in Tris buffer containing GSH and GSSG. The results showed that rvWF A3 was expressed successfully in E coli (strain DE3), accounting for 46% of total bacterial protein with its purity of over 95%. It was identified that rvWF A3 is capable to bind collagen and inhibit the wild vWF binding to collagen by competition. It is concluded that rvWF A3 fragment might be an effective antithrombotic agent for preventing arterial thrombosis.