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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。应用RT PCR技术 ,从分泌SZ 2 1单克隆抗体的杂交瘤细胞中克隆出VH 和VL 可变区基因 ,并构建成SZ 2 1单链抗体 (SZ 2 1ScFv)。将该单链抗体基因亚克隆到高效表达质粒pET2 0b ,得到pET2 0b 2 1ScFv。通过IPTG诱导在大肠杆菌EcoliBL2 1(DE3)PlysS中表达了功能分子。表达产物主要以包涵体形式存在 ,表达量占菌体蛋白的 2 1%。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重折叠等一系列的变性和复性过程 ,获得具有生物活性的高纯度单链抗体片段。ELISA和Westernblot证实该融合蛋白保留了与亲本抗体同样的血小板结合特性。SZ 2 1单链抗体体外能够抑制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 0mg/L时达到最大抑制率。流式细胞仪检测证实该单链抗体能与内皮细胞反应。该单链抗体有望用于血栓性疾病的治疗。
Monoclonal antibody SZ21 capable of specifically binding to platelet membrane glycoprotein IIIa is constructed as a single-chain antibody. The VH and VL variable region genes were cloned from the hybridoma cells secreting SZ21 monoclonal antibody by RT-PCR and constructed into SZ21 scFv. This single-chain antibody gene was subcloned into the highly efficient expression plasmid pET20b to give pET20b21ScFv. Functional molecules were expressed in E. coli Ecoli BL21 (DE3) PlysS induced by IPTG. The expression product mainly exists in the form of inclusion bodies, and the expression amount accounts for 21% of the bacterial protein. A series of degeneration and refolding processes of inclusion body, affinity purification of Ni NTA resin and in vitro refolding of protein were carried out to obtain bioactive high purity single chain antibody fragment. ELISA and Western blot confirmed that the fusion protein retained the same platelet binding properties as the parent antibody. SZ 2 1 single chain antibody in vitro can inhibit ADP-induced platelet aggregation, its inhibitory capacity in a dose-dependent manner, at a concentration of 20mg / L to achieve the maximum inhibition rate. Flow cytometry confirmed that the single-chain antibody reacts with endothelial cells. The single chain antibody is expected to be used in the treatment of thrombotic diseases.