抗血小板糖蛋白VI单链抗体的制备和功能研究  

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

作　　者：余自强[1] 董宁征[1] 白霞[1] 戴兰[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：《现代免疫学》2006年第1期41-47,共7页Current Immunology

基　　金：国家自然科学基金项目编号:30170915

摘　　要：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。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 SZllS. After ligation into the vector pET20b（＋）, expression plasmid pET20b（＋）-SZllSscFv 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 SZllSscFv reserved the binging activity to platelets identified by ELISA and flow cytom-etry. SZllSscFv was able to inhibit fibrillar collagen- and convulxin-induced platelet aggregation in a dose-de-pendent manner with maximum inhibition rate 84.3% 5：5.6% and 50.3% 5：15.5% re- spectively, and also SZ118scFv blocked the platelet adhesion to the fibrillar collagen under high shear rate with 68.3% inhibition. These data in-dicated that SZllSscFv 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.

关 键 词：糖蛋白VI 胶原 单链抗体 血小板 

分 类 号：R392.12[医药卫生—免疫学]