噬菌体展示CD28、CD137及生物活性测定  

Establish a high capability pharmic screening platform-Phage display CD28,CD137

作　　者：李娜[1] 张利宁[1] 高尚先[2] 王群[1] 刘兴田[1] 朱法良[1] 郭春[1] 

机构地区：[1]山东大学医学院免疫学研究所,济南250012 [2]中国药品生物制品检定所,北京100050

出　　处：《药物分析杂志》2005年第9期1106-1110,共5页Chinese Journal of Pharmaceutical Analysis

基　　金：国家自然科学基金(30371352;30271247)

摘　　要：目的:以噬菌体展示具有天然结构的人 CD28、CD137分子胞外区,检测其抗原性和生物活性,为 CD28、CD137拮抗性类肽药物的筛选奠定基础。方法:构建 CD28、CD137噬菌粒表达载体 pComb3H-CD28、pComb3H-CD137,以噬菌体展示系统展示 CD28、CD137分子胞外区。以 ELISA、流式细胞仪检测噬菌体展示 CD28的抗原性和与 CD80的结合力,ELISA 检测噬菌体展示 CD137的抗原性,MTT 法检测噬菌体展示 CD28、CD137的生物活性。结果:成功构建重组噬菌粒载体 pComb3H-CD28、pComK3H-CD137,并以噬菌体展示系统展示 CD28、CD137分子。ELISA 结果显示,噬菌体展示 CD28可与抗 CD28抗体特异性结合,也可与 CD80特异性结合;FCM 结果显示,噬菌体展示 CD28可与 Jurkat 细胞表面 CD28竞争结合抗 CD28抗体,也可与 Raji 细胞表面 CD80结合。ELISA 结果显示,噬菌体展示 CD137可与抗 CD137抗体特异性结合。体外生物活性实验显示,噬菌体展示 CD28、CD137可抑制抗 CD28抗体、抗 CD137抗体刺激的人外周血淋巴细胞增殖反应。结论:利用噬菌体展示系统成功展示 CD28、CD137分子,噬菌体展示 CD28、CD137具有抗原性及生物活性,为以 CD28、CD137为靶点体外高通量筛选CD28、CD137拮抗性类肽奠定基础。Objective：Using phage display technique to display human CD28,CD137 on phage and detect its antigenicity and bioactivity. Methods： Construct recombinant phagemid vector pComb3H- CD28 and pComb3H - CD137. Display CD28 and CD137 on phage surface. The antigenicity and bioactivity of CD28 and CD137 displayed on phage was detected by ELISA, FCM and inhibition assay of lymphocyte proliferation（ MTT）. Results ：The recombinant phagemid vector pComb3H - CD28 and pComb3H - CD137 was successfully constructed. CD28 and CD137 was displayed on phage. ELISA detection showed that phage -displayed CD28 could bind to CD28Ab and CD80; FCM detection indicated that phage - displayed CD28 could competitively bind to CD28Ab with CD28 on the surface of Jurkat cell and could also bind to Raji cell surface CD80, these data demonstrated that phage - displayed CD28 not only possesses antigenicity but also possesses its native structure binding to its ligand. ELISA detection showed that phage - displayed CD137 could bind to CD137Ab, it has CD137 antigenicity. In further, MTT inhibition assay showed that lymphocytes proliferation stimulated by CD28Ab, CD137Ab could be inhibited by phage - displayed CD28 ,CD137 respectively. Conclusion：The studies demonstrate that phage display system can display human CD28 extra - cellular domain homodimer with antigenicity and bioactivity.

关 键 词：CD28 CD137 噬菌体展示 抗CD28抗体 生物活性测定 ELISA检测 流式细胞仪检测 淋巴细胞增殖反应 体外生物活性 特异性结合 

分 类 号：R392.11[医药卫生—免疫学] R392-33[医药卫生—基础医学]