抗血小板糖蛋白VI单克隆抗体的制备及其功能研究  

Preparation and characterization of a monoclonal antibody directed against platelet glycoprotein VI

作　　者：余自强[1] 董宁征[1] 白霞[1] 沈飞[1] 季顺东[1] 邵波静[1] 阮长耿[1] 

机构地区：[1]苏州大学附属第一医院江苏省血液研究所,苏州215006

出　　处：《中国免疫学杂志》2006年第6期545-550,共6页Chinese Journal of Immunology

基　　金：江苏省"135工程"重点学科基金资助(SK200206)

摘　　要：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 develop monoclonal antibody（McAb） against platelet glycoprotein Ⅵ （GPⅥ） and determine its ability to inhibit platelet adhesion and platelet aggregation in vitro. Methods：The expression vector pET-20b （ ＋ ）-GPⅥ was constructed and the recombinent protein of GPⅥ extracellular domain（rGPⅥ） was expressed in E. coli BI21 （DE3） PlysS. After immunizing mice with rGPⅥ, a McAb against GPⅥ was developed by means of fusing mice spleen cells with myeloma cells and screening the hybridoma cells with HAT culture medium. The inhibitory effects of the McAb on platelet aggregation was measured. The platelet adhesion on surface of fibrillar collagen under high shear rate condition was observed by parallel plate flow chamber with McAb. Results： The rGPⅥ was successfully expressed in E. coli, which be identified by anti-Penta-His antibody and anti- GPⅥ polyclonal antibody. A McAb against GPⅥ, SZ118, was＇developed, which could＇react with platelet specifically. The collagen- and convulxin-induced platelet aggregation were markedly inhibited by SZ118, but less effect on ADP-induced platelet aggregation. Platelet adhesion on the surface of fibril- lar collagen under high shear rate condition was also impaired by SZ118. ConcIusion：The prepared McAb SZ118 was able to bind to platelets exclusively, and exhibited inhibitory activity on the platelet aggregation induced by fibrillar collagen and convulxin and the platelet adhesion to collagen.

关 键 词：血小板 糖蛋白Ⅵ 胶原 单克隆抗体 

分 类 号：R392.12[医药卫生—免疫学]