中和抗肾小球基底膜抗体的单链抗体的筛选与鉴定  

Screening and identification of neutralizated single-chain antibody of anti-glomerular basement membrane antibody

作　　者：肖静[1] 刘章锁[1] 王沛[1] 黄留玉[2] 宋宏彬[2] 赵明辉[3] 

机构地区：[1]郑州大学第一附属医院肾内科,450052 [2]北京军事医学科学院 [3]北京大学第一医院

出　　处：《中华微生物学和免疫学杂志》2010年第9期791-794,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家卫生部资助项目（WKJ2007-2-033）;河南省医学科技攻关重大项目（200701006）

摘　　要：目的 制备人抗肾小球基底膜（GBM）抗体的特异性人源化单链可变区抗体.方法 采用噬菌体表面展示技术,获得一个与人抗GBM抗体结合活性较强的单链可变区抗体片段的阳性克隆,并对该克隆进行DNA序列测定分析.结果 对噬菌体单链可变区抗体库经过3轮筛选后,与第1轮相比富集了137倍.噬菌体抗体与人抗GBM抗体的结合活性其中有35株克隆ELISA的吸光度较高.对这些噬菌体抗体进行交叉反应后,确定其中有10株交叉反应较弱.确定1株（C31）阳性克隆提取质粒,进行DNA序列测定,大小为750 bp,并符合人源化单链可变区抗体的序列结构.结论 应用噬菌体展示技术成功获得人-抗GBM抗体的单链可变区抗体基因,为临床上治疗Goodpasture综合征奠定实验基础.Objective To screen a human single-chain variable fragments（scFv）against antiGBM antibody.Methods Using phage display technique,the phage antibody library was panned by antiglomerular basement membrane（GBM）antibody which was coated in a micro-titer plate,one clone was found to have high affinity to anti-GBM antibody.The DNA sequence of the positive clone was determined.Results Along with the increase of rounds anti-GBM antibody specific phage antibody was highly enriched and screening efficiency was increased 137 folds than the firest round.ELISA and competition inhibition assay showed that the scFv had a specific combination character with anti-GBM antibody.DNA sequencing confirmed that the whole gene of scFv was 750 bp,and in accordance with humanized single-chain variable region antibody sequence structure.Conclusion The results suggested that the scFv fragment to anti-GBM antibody could be successfully selected by recombinant phage antibody technique,which will laid an experimental foundation for further research of the therapy of Goodpasture syndrome.

关 键 词：噬菌体展示技术 抗肾小球基底膜抗体 

分 类 号：R392.1[医药卫生—免疫学]