噬菌体展示法筛选抗人PTA1单克隆抗体结合肽  

Identification of the Peptides that Bind to PTA1 mAb(LeoA1) by Phage Displayed Library

作　　者：杨琨[1] 金伯泉[1] 贾卫[1] 张新海[1] 欧阳为明[1] 朱勇[1] 刘雪松[1] 张晓光[2] 聂晓燕[2] 

机构地区：[1]第四军医大学免疫学教研室,陕西西安710032 [2]第四军医大学生物化学与分子生物学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2001年第4期328-331,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助;No.39980006;30030130

摘　　要：目的从噬菌体展示文库中筛选能与抗人PTA1单克隆抗体(mAbs)特异性结合的短肽。方法采用protein-A亲和层析法纯化系列抗人PTA1mAbs，通过流式细胞术检测筛选出能够阻断PTA1-Fc融合蛋白与其天然配体结合的mAb，经过三轮亲和筛选，从噬菌体随机12肽库中筛选可特异性结合mAb的重组噬菌体阳性克隆，进而对这些克隆的短肽序列进行测定和分析。结果确定了能够阻断PTA1-Fc融合蛋白与其天然配体结合的mAb为LeoA1，得到了13个具有特异性结合LeoA1的重组噬菌体阳性克隆，序列测定结果发现，这些可结合LeoA1的短肽，有较保守而集中的模式序列，即WPXHHX序列。结论这些具有保守模式序列的短肽，有可能模拟PTA1分子的功能表位，成为其天然配体拮抗剂的候选肽段。此外，PTA1分子与其配体结合的功能表位的确定，也为今后寻找天然配体和进一步深入研究PTA1分子的结构和功能提供了实验依据。Aim To screen peptides binding specifically to anti-human PTA1mAbs from a random twelve-peptide phage-disp-layed library. Methods Series of PTA1mAbs(LeoA1、1B11、C9、2D1、2E9、2G8、2H2 and E8)were purified using protein-A affinity column. PTA1mAbs which could bind PTA1-Fc fusion protein binding to its ligand were confirmed by flow cytometry, and then used as target to screen phage library. After three rounds of affinity screening, the peptide sequences of positive phage clones were determined and analyzed. Results LeoA1 could block PTA1-Fc fusion protein binding to its ligand. 13 phages which could bind specifically to LeoA1 were isolated from phage library and further confirmed by ELISA. Conserved motifs were found among the sequences of the peptides. Conclusion It was shown that the conserved motifs were candidafe regions binding to PTA1 ligand,which is important to identify functional epitopes for seeking ligand of PTA1 and further investigation of biological function of PTA1.

关 键 词：PTA1 单克隆抗体 抗原表位 噬菌体展示 随机短肽库 

分 类 号：Q782[生物学—分子生物学] R392.11[医药卫生—免疫学]