抗血小板糖蛋白VI单链抗体的制备和功能研究

将抗血小板膜表面糖蛋白VI(GPVI)单克隆抗体SZ118构建成单链抗体,以研究开发新型抗血栓药物。采用RT-PCR技术,从分泌SZ118杂交瘤细胞中克隆该抗体的重链可变区(VH)和轻链可变区(VL)基因。经测序鉴定后构建表达质粒pET20b(+)-SZ118scFv,并体外表达SZ118单链抗体(SZ118scFv)。同时测定了SZ118scFv对血小板黏附和聚集功能的影响。结果表明成功克隆了SZ118 VH和VL基因并正确构建了SZ118scFv表达质粒。复性后SZ118scFv保留源抗体与血小板结合能力,显著抑制纤维状胶原和Convulxin诱导的血小板聚集,且呈剂量依赖性,最大抑制率分别为(84.3%±5.6%)和(50.3%±15.5%);SZ118scFv明显抑制高剪切力条件下血小板与纤维状胶原的黏附,抑制率达68.3%。结果提示,成功制备了SZ118scFv,SZ118scFv可阻断血小板与胶原的相互作用,具有潜在的实际应用价值。

Preparation and characterization of a single chain antibody of McAb SZ118 against platelet glycoprotein VI

To develop a potential antithrombotic reagent,a single chain antibody(scFv) from monoclonal antibody(McAb)SZ118 against platelet glycoprotein VI was prepared.The gene encoding for the light-and heavy-chain variable regions had been cloned by RT-PCR from a murine hybridoma that produced SZ118.After ligation into the vector pET20b(+),expression plasmid pET20b(+)-SZ118scFv was contructed,and SZ118scFv fragment was ex-pressed in E.coli.SZ118scFv was characterized in platelet adhesion and aggregation to collagen.The results showed that the purified SZ118scFv reserved the binging activity to platelets identified by ELISA and flow cytom-etry.SZ118scFv was able to inhibit fibrillar collagen-and convulxin-induced platelet aggregation in a dose-de-pendent manner with maximum inhibition rate 84.3%±5.6% and 50.3%±15.5% respectively,and also SZ118scFv blocked the platelet adhesion to the fibrillar collagen under high shear rate with 68.3% inhibition.These data in-dicated that SZ118scFv was successfully prepared,which could interfere with the interaction between GPVI and collagen and was in the hope of the treatment of for treating thrombotic diseases.