摘要
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 acquire recombinant protein of GPIbα（H1-V289）,which is located at the N terminal of platelet membrane glycoprotein Ibα,for further studies on its biological function in thrombosis and hemostasis.Methods The prokaryotic expression vector pQE30-GPIbα（H1-V289） was constructed using the template of plasmid pCMV3（coding GPIbα H1-V289）,and then transfected into E coli M15,following the induction of IPTG.The recombinant protein was purified with Ni-NTA agarose column by gradient pH value.The purity and immune activity of purified products were identified with SDS-PAGE and Western blot respectively.The effect of rGPIbα（H1-V289） on the platelet aggregation function was detected with plate aggregation assay.Results The result showed that we successfully got the protein of rGPIbα（H1-V289） with the high purity.The concentration was 0.6 mg/ml.The recombinant fragment could react with monoclonal antibody SZ-2 against GPIbα by Western blot.It could inhibit the platelet aggregation induced by ristocetin but not ADP.Conclusion The recombinant protein exhibits good immune activity and biology activity,which could establish the experimental foundation for further research on antithrombotic drugs.
出处
《苏州大学学报（医学版）》
CAS
北大核心
2010年第6期1151-1155,共5页
Suzhou University Journal of Medical Science
基金
国家自然科学基金资助项目(30670904)
