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目的 :进一步研究血栓形成的机制 ,开发抗血栓药物。方法 :应用基因重组技术在大肠杆菌中表达人vWF -A1区蛋白 ,经过纯化、复性 ,获得重组蛋白 (rvWF -A1) ,同时用流式细胞术检测rvWF -A1与血小板膜糖蛋白血小板膜糖蛋白 (glycoprotein ,GP)Ib的结合能力 ,应用血小板聚集仪测定rvWF -A1对瑞斯托霉素 (ristocetin)诱导的血小板聚集抑制作用。结果 :重组表达载体pQE -3 1-vWF -A1在大肠杆菌M15中得到高效表达 ,表达的重组蛋白量占菌体总蛋白的 3 0 % ,Ni -NTAagrose柱纯化后 ,其纯度为 95% ,经复性的rvWF -A1蛋白具有良好的生物学活性。它可与血小板模糖蛋白血小板膜糖蛋白GPIb结合 ,阳性率为 78 6% ;它可以抑制ristocetin诱导的血小板聚集 ,抑制率为 84 7%。结论 :在原核细胞中可以成功地高效表达人vWF -A1区蛋白 ,该重组蛋白有可能开发为有效的抗血栓药物
Objective: To further study the mechanism of thrombosis and develop antithrombotic drugs. Methods: Recombinant human vWF-A1 protein was expressed in Escherichia coli by recombinant DNA technology. After purification and refolding, the recombinant protein (rvWF-A1) was obtained. The expression of rvWF-A1 and platelet membrane glycoprotein Glycoprotein (GP) Ib binding capacity, inhibition of ristocetin-induced platelet aggregation by rvWF-A1 was measured using a platelet aggregation assay. Results: The recombinant plasmid pQE-3 1-vWF-A1 was highly expressed in E. coli M15. The expressed recombinant protein accounted for 30% of the total bacterial proteins. The purified recombinant protein was purified by Ni-NTA agrose and its purity was 95% Refolded rvWF-A1 protein has good biological activity. It can be combined with platelet glycoprotein platelet glycoprotein GPIb, the positive rate was 78 6%; it can inhibit ristocetin-induced platelet aggregation, the inhibition rate was 84 7%. CONCLUSION: Human vWF-A1 region protein can be efficiently and efficiently expressed in prokaryotic cells, and it is possible that the recombinant protein is developed as a potent antithrombotic agent