Identification, Characterization, Cloning, and Cross-Reactivity of Zan b 2, a Novel Pepper Allergen of 11S Legumin.

Abstract

Protein from Sichuan peppers can elicit mild to severe allergic reactions. However, little is known about their allergenic proteins. We aimed to isolate, identify, clone, and characterize Sichuan pepper allergens and to determine its allergenicity and cross-reactivities. Sichuan pepper seed proteins were extracted and then analyzed by SDS-PAGE. Western blotting was performed with sera from Sichuan pepper-allergic individuals. Proteins of interest were purified using hydrophobic interaction chromatography and gel filtration and further analyzed by analytical ultracentrifugation, circular dichroism spectroscopy, and mass spectrometry (MS). Their coding region was amplified in the genome. IgE reactivity and cross-reactivity of allergens were evaluated by dot blot, enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. Western blot showed IgE binding to a 55 kDa protein. This protein was homologous to the citrus proteins and has high stability and a sheet structure. Four DNA sequences were cloned. Six patients' sera (60%) showed specific IgE reactivity to this purified 11S protein, which was proved to have cross-reactivation with extracts of cashew nuts, pistachios, and citrus seeds. A novel allergen in Sichuan pepper seeds, Zan b 2, which belongs to the 11S globulin family, was isolated and identified. Its cross-reactivity with cashew nuts, pistachios, and citrus seeds was demonstrated.