人源性抗血小板膜糖蛋白Ⅱb/Ⅲa Fab噬菌体抗体库的筛选与鉴定  

Screening and Identification of Human Fab Phage Display Library Against Platelet Membrane Glycoprotein Ⅱb/Ⅲa

作　　者：崔宇杰[1] 董宁征[1] 庞爱明[1] 白霞[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,江苏苏州215006

出　　处：《苏州大学学报（医学版）》2008年第5期696-698,732,共4页Suzhou University Journal of Medical Science

基　　金：江苏省基础研究计划(自然科学基金)资助项目(BK2005036)

摘　　要：目的从已构建的人源性抗血小板膜糖蛋白(glycoprotein,GP)Ⅱb/ⅢaFab噬菌体抗体库中筛选人源性抗血小板膜GPⅡb/ⅢaFab抗体,并对特异性阳性克隆进行鉴定。方法以表达GPⅡb/Ⅲa的CHO123细胞包板,对人源性抗血小板膜GPⅡb/ⅢaFab噬菌体抗体库进行富集筛选,采用细胞ELISA法筛选特异性阳性克隆,用Western blot法鉴定表达蛋白与GPⅡb/Ⅲa结合的特异性,将阳性克隆进行扩增、测序。结果以CHO123细胞富集淘筛3轮,并用ELISA法检测到2个高亲合力的特异性识别血小板膜GPⅡb/Ⅲa的克隆,Westernblot鉴定结果显示在阳性克隆的培养上清和细菌冻融上清均有特异性条带出现。对核苷酸序列进行同源性分析,证实这两个克隆轻链基因与人免疫球蛋白κ轻链的同源性分别高达97%和98%,重链基因与人免疫球蛋白Fd段的同源性分别高达94%和93%。结论利用噬菌体抗体库技术,成功地从人源性抗血小板膜GPⅡb/ⅢaFab抗体库中筛选出抗血小板膜GPⅡb/Ⅲa的特异性阳性克隆,该克隆具有较高的特异性,其克隆基因符合抗体可变区结构特点。Objective To screen the antibodies against platelet membrane glycoprotein Ⅱ b/Ⅲa （GPⅡ b/Ⅲa） from the constructed human Fab fragment phage display library and to identify specific positive clones. Methods For screening and enrichment, ELISA plate was coated with Chinese hamster ovary （CHO） cells expressing GP Ⅱ b/Ⅲa, CHO123 cell line. Specific positive clones by cyto-ELISA. The binding specificity of its protein to GPⅡ b/Ⅲa was detected by Western blot. Amplified and sequenced specific positive clone genes. Results After three rounds of panning with CHO123,phage antibodies against GPⅡ b/Ⅲa were enriched specifically. Two positive phage antibodies that were highly specific for GPⅡ b/Ⅲa were verified by phage-ELISA detection. Western-blot showed that there was specific band in culture supernatant and frozen-thawn supernatant of positive clone. Sequence of light chain had the homology of 97% with s： region and Fd fragmen having the homology of 94% with human immunoglobulin. Conclusion The positive clones specifically binding with GPⅡ b/Ⅲa have been selected by phage display.The positive clones were highly specific and the sequence was consistent with classical antibody variable region structure.

关 键 词：血小板膜糖蛋白 Ⅱb/Ⅲa 噬菌体抗体库 筛选 鉴定 

分 类 号：R392.11[医药卫生—免疫学]