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目的:制备抗血小板糖蛋白VI(GPVI)单克隆抗体,观察其在体外抗血小板黏附和聚集功能。方法:采用基因重组技术体外表达血小板糖蛋白VI胞外区重组蛋白(rGPVI)。以rGPVI免疫小鼠,经细胞融合及筛选后制备抗GPVI单克隆抗体。采用血小板聚集实验观察该单抗对胶原、Convu lxin及ADP诱导的血小板聚集的影响;利用平行板流动小室技术研究在高剪切力条件下该单抗对血小板在胶原表面黏附的抑制效果。结果:正确构建了rGPVI表达载体pET-20b(+)-GPVI,rGPVI在原核细胞中有效表达。rGPVI能够被抗Penta-H is单抗和抗GPVI多抗识别。制备的抗GPVI单克隆抗体SZ118能够识别rGPVI,并与血小板有特异的结合能力。SZ118能明显抑制纤维状胶原和Convu lxin诱导的血小板聚集,呈抗体剂量依赖性;对ADP诱导的血小板聚集无明显影响。血小板黏附实验表明,SZ118能够明显阻断在高剪切力条件下血小板与纤维状胶原表面的黏附。结论:成功制备抗GPVI单克隆抗体SZ118,该抗体与血小板有良好的结合能力,显著抑制胶原诱导的血小板聚集并明显降低血小板与胶原的黏附反应。
Objective: To prepare anti-platelet glycoprotein VI (GPVI) monoclonal antibody and observe its anti-platelet adhesion and aggregation function in vitro. Methods: The recombinant protein of extracellular domain of platelet glycoprotein VI (rGPVI) was expressed in vitro by gene recombination technique. Mice were immunized with rGPVI and monoclonal anti-GPVI antibodies were prepared by cell fusion and screening. The platelet aggregation test was used to observe the effect of the monoclonal antibody on collagen, Convu lxin and ADP-induced platelet aggregation. Parallel plate flow cell technique was used to study the inhibitory effect of the monoclonal antibody on platelet adhesion to collagen surface under high shear stress. Results: The rGPVI expression vector pET-20b (+) - GPVI was correctly constructed and rGPVI was efficiently expressed in prokaryotic cells. The rGPVI can be recognized by anti-Penta-His monoclonal antibody and anti-GPVI polyclonal antibody. The prepared anti-GPVI monoclonal antibody SZ118 can recognize rGPVI and has specific binding ability to platelets. SZ118 significantly inhibited platelet aggregation induced by fibrillar collagen and Convu lxin in a dose-dependent manner and had no significant effect on ADP-induced platelet aggregation. Platelet adhesion experiments showed that SZ118 could obviously block the adhesion of platelets to fibrillar collagen under high shear stress. CONCLUSIONS: The anti-GPVI monoclonal antibody SZ118 has been successfully prepared. The antibody has good binding ability with platelets, significantly inhibits collagen-induced platelet aggregation and significantly reduces platelet adhesion to collagen.