苏州大学附属第一医院江苏省血液研究所.pdfVIP

戴克胜等　抗血小板膜糖蛋白单克隆抗体 SZ2 单链抗体的构建、表达及功能研究　第 4 期 2·43 · 抗血小板膜糖蛋白单克隆抗体 SZ2 单链抗体的构建、表达及功能研究 ① ① ( ) 戴克胜 　祝怀平 　阮长耿 　 苏州大学附属第一医院江苏省血液研究所 ,苏州 215006 　　中国图书分类号 　R39211 　　文献标识码 　A 　　文章编号 　1000484X( 2003) [摘 　要] 　目的 : 降低鼠源性抗体的免疫原性及分子量 ,为其进一步研究、应用奠定基础。方法 :应用 RTPCR 技术 ,克隆 SZ2 重链、轻链可变基因。应用基因重组技术构建 SZ2 单链抗体表达载体pET222scFv ,导入大肠杆菌 BL21 (DE3) Plys ,诱导表 达。流式细胞术、ELISA 、Western blot 检测 SZ2 scFv 与血小板结合能力 ,瑞斯托霉素、凝血酶和 ADP 诱导血小板聚集试验对表 达产物功能进行研究。结果 :克隆基因序列符合小鼠轻、重链可变区基因特征 ;pET222scFv 表达质粒拼接正确 ;表达产物以包 涵体形式为主 ,表达量占菌体总蛋白的 25 % ;纯化、复性后证明表达产物具有与血小板结合的活性 ,并可抑制瑞斯托霉素、凝 血酶诱导的血小板聚集。结论 :成功表达了 SZ2 单链抗体 ,该小分子抗体具有与血小板结合的活性 ,并可抑制瑞斯托霉素、凝 血酶诱导的血小板聚集。 [ 关键词] 　单链抗体 ;单克隆抗体 ;血小板 Production and expression of a single chain antibody against platelet in E. coli DAI KeSheng , ZHU HuaiPing , RUAN ChangGeng . The First Affiliated Hospital of Suzhou University ,Jiangsu Institute of Hematology , Suzhou 215006 , China ( ) [ Abstract ] 　Objective :To express the recombinant single chain Fv scFv in E. coli and reduce immunogenicity and molecular weight of a monoclonal antibody specific for human platelet. Methods :The variable regions of the heavy and light chains of plateletspecific antibody SZ2 were amplified by reverse transcription and polymerase chain reaction. VH and VL gene segments were cloned into pUCTm and joined together with a (gly ser) linker. The resulting scFv was expressed in PET expression system. The expressed recombinant protein was characterized by its 4 3 size on SDSPAGE ,by Western blot ,by flow cytometry and its functions. Results :The VH and VL genes were homologous with the published gene sequences of mouse antibody variable region. The recombinant scFv was expressed mostly in the form of inc