【摘要】 目的　将能与血小板膜糖蛋白Ⅲa特异结合的单克隆抗体SZ 2 1构建成单链抗体 (SZ 2 1ScFv) ,为其临床应用奠定基础。方法　构建SZ 2 1ScFv基因的质粒pET2 0b 2 1ScFv ,在大肠杆菌EcoliBL2 1(DE3)PlysS中表达了功能分子。表达产物经过包涵体的溶解、Ni NTA树脂亲和吸附纯化和蛋白质的体外复性过程 ,获得具有生物活性的高纯度单链抗体片段。结果　体外实验证实表达量占菌体蛋白的 2 1%。SZ 2 1单链抗体能够抑制ADP诱导的血小板聚集 ,其抑制能力呈剂量依赖性 ,在浓度为2 0 μg/ml时达到最大抑制率。流式细胞术检测证实该单链抗体能与内皮细胞反应 ,同时也可以抑制纤维蛋白原与血小板的结合。结论　表达的单链抗体具有抑制血小板聚集和抑制纤维蛋白原与血小板结合的能力 ,有望用于血栓性疾病的治疗更多还原

【Abstract】 Objective To prepare a single chain antibody (ScFv) of mAb SZ-21 against platelet GPⅢa for its future clinical application. Methods The expression vector pET20b-SZ-21ScFv was constructed and the fusion protein was expressed in E. coli BL21(DE3)PlysS. The activated fusion protein was obtained after a series of purification steps, including cell breakage, inclusion body solubilization, His-bind resin affinity chromatography and protein refolding. Results The fusion protein yields were up to 21% of the total amount of bacteria protein. The ScFv fragment could inhibit ADP-induced platelets aggregation in a dose-dependent manner in vitro and the maximal inhibition rate was obtained at a concentration of 20 μg/ml. It also reacted with endothelial cells as detected by flow cytometry. Moreover, the ScFv fragment was able to inhibit the binding of fibrinogen to platelet. Conclusion The SZ-21ScFv fragment had the activity to inhibit platelets aggregation and the binding of fibrinogen to platelet, being potentially useful for the treatment of thrombotic diseases.更多还原