人vWF-A1区蛋白的表达及其对血小板聚集的抑制作用

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-31-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30%,Ni-NTA agrose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。

Expression of von Willebrand factor-A1 domain in E coli and the inhibitory effect on platelet aggregation

<b>AIM:</b> To further investagate the mechanism of thrombus formation and develop a new remedy of anti-thrombus formation. <b>METHODS:</b> The amplified DNA fragment of vWF-A1 domain was inserted into expression vector with 6×his taq (pQE-31), the recombinant expression vector was transformed into E coli (strain M15) and induced by IPTG. The recombinant fragment, comprising residues 449-728 of mature vWF subunit, designate rvWF-A1. It was purified by Ni-NTA agarose column and renatured by Tris buffer containing GSH and GSSG. FACS and platelet aggregometer were employed to analyse the rvWF-A1 function of binding to platelet glycoprotein Ib and inhibiting ristocetin-induced platelet aggregation. <b>RESULTS:</b> The rvWF-A1 was expressed successfully in E coli, coming up to 30% of total bacterial protein. Its purify was over 95% through Ni-NTA agarose. It was identified to have ability to bind to GPIb, its biologic activity to inhibit ristocetin-induced platelet aggregation was observed, and the inhibitive rate was 84.7%. <b>CONCLUSION:</b> The above results indicated that high-level expression of rvWF-A1 was successfully achieved in E coli and rvWF-A1 may be an effective antithromotic agent in preventing thrombus formation.