抗血小板GP Ⅲa单克隆抗体SZ-21单链抗体片断的表达和特性

Expression and characterization of a single chain antibody fragment of mAb SZ-21 against platelet GPIIIa

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。

A single chain antibody (ScFv) from mAb SZ 21 against platelets GPIIIa has been produced for its clinical application. The genes encoding for the light and heavy chain variable regions(V H and V L) have been cloned by RT PCR from a murine hybridoma that produces SZ 21 monoclonal antibody and constructed a SZ 21ScFv fragment. ScFv gene was ligated into the highly expressed vector pET20b, the fusion protein was expressed in Eschrichia coli BL21(DE3)PlysS by IPTG induction. The recombinant protein was mostly in the form of inclusion bodies and yield was up to 21% of the total amount of bacteria protein . The pure fusion protein was obtained after a series of purification steps including cell breakage, inclusion body solubilization, His bind resin affinity chromatography and protein refolding. The ScFv fragment has the same binding activity to platelet as mAb SZ 21 identified by ELISA and Western blot. ADP induced platelet aggregation can be inhibited by the ScFv fragment in a dose dependent manner in vitro and the maximal inhibition rate was obtained at a concentration of 20mg/L. The ScFv fragment also reacts with endothelial cells by flow cytometry. In conclusion, the SZ 21ScFv fragment retained the binding ability and the biological activities of their murine counterparts, which could be useful in the treatment of thrombosis diseases.