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目的获得重组的血小板膜糖蛋白GPIbα的N端片段(1-289氨基酸),进一步研究其在血栓与止血过程中的生物功能。方法利用质粒pCMV3(编码GPIbαH1-V289)设计引物,构建pQE30-GPIbα(H1-V289)表达载体,转化大肠杆菌M15,IPTG诱导表达蛋白。用Ni-NTA琼脂糖层析柱不同pH值梯度淋洗法纯化蛋白,SDS-PAGE和Western blot鉴定纯化产品纯度和免疫学活性,并观察重组蛋白对瑞斯托霉素和ADP诱导血小板聚集的影响。结果成功获得纯度较高的重组GPIbα(H1-V289)蛋白,浓度为0.6 mg/ml。Western blot检测结果显示,抗GPIbα单抗SZ2能在34 kd区域显示条带。而且纯品能有效抑制瑞斯托霉素诱导的血小板聚集,对ADP诱导的反应无抑制作用。结论重组蛋白具有较好的免疫原性和生物活性,并可以大量获得,为开发抗血栓药物奠定了基础。
Objective To obtain the N-terminal fragment (1-289 amino acids) of the recombinant platelet membrane glycoprotein GPIbα and to further study its biological function in thrombosis and hemostasis. Methods The plasmid pCMV3 (encoding GPIbαH1-V289) was designed and used to construct pQE30-GPIbα (H1-V289) expression vector. The recombinant plasmid was transformed into E. coli M15 and induced by IPTG. The purified proteins were purified by Ni-NTA agarose column with different pH gradient elution. Purity and immunological activity of purified product were identified by SDS-PAGE and Western blot. The effects of recombinant protein on ristocetin and ADP-induced platelet aggregation influences. Results The highly purified recombinant GPIbα (H1-V289) protein was successfully obtained at a concentration of 0.6 mg / ml. Western blot results showed that anti-GPIbα mAb SZ2 showed a band in the 34 kd region. And pure can effectively inhibit ristocetin-induced platelet aggregation, no inhibition of the ADP-induced response. Conclusion The recombinant protein has good immunogenicity and biological activity, and can be obtained in large quantities, which lays the foundation for the development of antithrombotic drugs.