Identification and Bioinformatics Analysis of Malate Dehydrogenase and Pyruvate Kinase as Novel Allergens in Cicada Pupae

Cicada pupae are a traditional and promising food source, and it is crucial to evaluate the associated allergenic risks. In this study, 13 potential allergens were identified based on proteomics combined with immunoinformatics tools in cicada pupae. These allergens were produced as recombinant proteins, and their allergenicity was investigated. Malate dehydrogenase (MDH) and pyruvate kinase (PK) were finally identified as new allergens via immunoblot experiments. The secondary structures of the two new allergens predominantly consist of <i>α</i>-helices and <i>β</i>-sheets. Five IgE-binding epitopes of MDH and four of PK were identified. AA13-32, AA 297-306 in MDH, and AA124-132, 206-221 in PK exhibited strong IgE reactivity and were identified as the primary linear IgE-binding sites. The epitopes of MDH contain mainly α-helix and loop structures, whereas those of PK consist primarily of an α-helix. These findings provide a reference for developing diagnostic and immunotherapeutic strategies of cicada pupae allergy.