利用噬菌体随机9肽库筛选寻常型天疱疮抗原模拟表位

Selection of mimotopes of pemphigus vulgaris antigen from a phage-displayed random nonapeptide library

目的利用噬菌体随机9肽库筛选寻常型天疱疮抗原桥粒芯糖蛋白3（desmoglein，Dsg3）模拟表位，加深对寻常型天疱疮发病机制的认识。方法通过大肠杆菌表达Dsg3的胞外结构域1和2（extracellular domain，EC1-2）和谷胱甘肽转移酶（GST）的融合蛋白，从寻常型天疱疮患者血清中纯化与EC1-2特异结合的多克隆抗体，对噬菌体随机线九肽库和环九肽库进行亲和筛选，阳性噬菌体展示肽经ELISA和竞争性ELISA验证。结果经过两轮亲和筛选，与自身抗体结合的噬菌体明显富集，免疫筛查、验证后得到3个阳性噬菌体展示肽，ELISA检测显示它们与患者血清反应，而不与正常人血清反应，竞争性ELISA检测显示它们可以抑制寻常型天疱疮患者血清与重组蛋白EC1-2的结合。结论利用噬菌体随机9肽库筛选到3个与寻常性天疱疮密切相关的模拟表位。

Objective To screen the mimotopesofpemphigus vulgaris （PV） antigen, desmoglein3 （Dsg3） with a phage-displayed random nonapeptide library, so as to update the knowledge on the patho- genesis of PV. Methods Recombinant fusion protein of extracellular domain 1-2 （EC1-2） of Dsg3 and glutathione transferase was expressed by E.coli BL21, and used to purify polyclonal autoantibody binding to recombinant EC1-2 from the sera of patients with PV. Then, selected autoantibody was applied as a ligand for biopanning of a phage-displayed linear random nonapeptide library and circular random nonapeptide library. Monoclonal phages were selected by immunoscreening and tested with ELISA and competitive ELISA. Results After two rounds ofbiopanning, a population ofpeptide-displaying phages binding to autoantidody were highly enriched. Sixty individual phage clones selected by immunoscreening were further subjected to screening with ELISA and competitive ELISA. Finally, three positive phage clones were obtained. As shown by ELISA and competitive ELISA, they reacted with serum from patients with PV but not with that from normal human controls, and blocked the interaction between patients＇ sera and recombinant fusion protein of EC1-2. Conclusion Three mimotopes closely associated with PV antigen were successfully selected from a phage-displayed random nonapeptide library.