利用大型天然噬菌体抗体库筛选抗死亡受体5人源抗体  

Screening of anti-human death receptor 5 antibody from a large natural phage antibody library

作　　者：蒋效[1] 何立东[2] 王欲晓[2] 丁立[2] 吴成林[2] 熊丽娟[2] 周丽君[2] 王育红[1] 

机构地区：[1]第二军医大学海军临床医学院普通外科 [2]海军总医院中心实验科,北京100048

出　　处：《细胞与分子免疫学杂志》2015年第6期833-837,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家科学自然基金(31470897;81172119)

摘　　要：目的从大容量天然噬菌体抗体库中筛选抗人死亡受体5(DR5)单链抗体(sc Fv)并对其抗原结合活性进行鉴定。方法以前期真核表达纯化的DR5-Fc蛋白包被筛选管,从自主构建的大容量天然噬菌体抗体库中,通过4轮筛选过程获得噬菌体抗体,采用免疫细胞化学染色、ELISA鉴定其抗原结合活性;DNA指纹图谱分析和序列测定确定其抗体类型。结果经过4轮筛选,获得26株与DR5特异性结合的阳性克隆,DNA指纹分析有4种不同的可变区片段;经过基因测序,分析可变区基因的亚群,并将其制备成可溶性的抗体,进一步用免疫细胞化学技术证实所获得的抗体可特异性结合SW480细胞的DR5蛋白。结论利用噬菌体抗体库技术成功获得了特异性抗DR5的人源性sc Fv。Objective To clone anti-human death receptor 5 （DRS） single-chain variable fragments （scFvs） from a large natural phage antibody library and identify the antigen-binding activity. Methods Phage antibodies against human DR5 were screened from a large natural phage antibody library constructed in our laboratory by four rounds of panning process using eukaryotically expressed DRS-Fc protein. The antigen-binding activity of the obtained scFvs was determined by immunocytochemistry and ELISA, and then the scFVs were analyzed by DNA fingerprinting and sequence analysis. Results After 4 rounds of panning, we obtained 26 positive clones with specific binding ability to DR5 protein. DNA fingerprinting and sequence analysis showed that there were four different variable fragments that were derived from different gene families. Soluble scFv fragments were prepared and then confirmed to bind specifically to DR5 protein in SW480 cells by immunochemistry. Conclusion Specific anti-human DR5 scFvs has been obtained from a large phage antibody library.

关 键 词：抗死亡受体5抗体 人源单链抗体 大容量噬菌体抗体库 

分 类 号：R73-362[医药卫生—肿瘤] R392.11[医药卫生—临床医学]