关键词: 噬菌体随机肽库, 寻常性天疱疮抗原, 模拟表位

Abstract: Objective To screen the mimotopes of pemphigus vulgaris （PV） antigen, desmoglein3 （Dsg3） with a phage-displayed random nonapeptide library, so as to update the knowledge on the pathogenesis 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 of biopanning, a population of peptide-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.

Key words: phage display peptide library, pemphigus vulgaris antigen, mimotopes