Localization of G3A1b Destroyed by Heat Treatment and Identification of Allergenic Amino Acids

Abstract

The G3 subunit is a key allergenic component of glycinin, a major soybean protein. This study utilized molecular cloning and recombinant phage construction to investigate antigenic sites in the G3 subunit that are denatured during heat treatment. Using indirect ELISA, the G3A1b-3–B-II fragment was identified as the denatured antigenic site, further localized to the sequence ²³⁶RQIVRKLQGENEEEEKGAIVTVKGGLSV²⁶³ through three rounds of screening. Alanine-scanning mutagenesis revealed that residues V255, T256, V257, G259, and L261 are critical for the binding of synthetic peptide P3 (²⁵¹KGAIVTVKGGLSV²⁶³) to IgG and IgE. These findings provide a refined understanding of the amino acid residues that influence glycinin allergenicity. This research lays the groundwork for reducing or eliminating soybean allergenicity through targeted amino acid substitutions, advanced biological breeding techniques, and other interventions. This method overcomes the defect that heat treatment cannot completely eliminate the allergenicity of glycinin.