Abstract: To identify the critical amino acids in the linear immunoglobulin (IgE)-binding epitopes of Jug r 1, a major English walnut allergen and to evaluate the accuracy and efficiency of two immunoinformatics methods in predicting these amino acids, the present study used the immunoinformatics methods to predict the key amino acids in the three linear epitopes of Jug r 1 and identified the amino acids by alanine scanning mutagenesis. Then a series of mutant peptides were synthesized by a solid phase method for the characterization of the epitopes and critical amino acids. The IgE-binding epitopes were immunolabeled with individual sera from walnut-allergic patients in China as probes by enzyme linked immunosorbent assay (ELISA). The results showed that Leu4, Leu7, Leu8, Ile20, Thr21, Ile22, Ile127, Ser129 and Gln130 were the critical amino acids in the IgE-binding epitopes. The combination of the two immunoinformatics gave 100% predication accuracy, but the prediction efficiency was only 11%. Therefore, the prediction accuracy can be improved by the combined use of the immunoinformatics methods, but some critical amino acids may be neglected. Immunoinformatics combined with alanine scanning mutagenesis is an important approach to identify the critical amino acids in the epitopes.

Key words: English walnut; Jug r 1; critical amino acids; linear epitopes; immunoinformatics