Identification of β-conglycinin α' subunit antigenic epitopes destroyed by thermal treatments

Abstract

In the present study, phage display technology (PDT) was used to examine β-conglycinin α' subunit epitopes destroyed by heat treatments. The overlapping gene fragments of the α' subunits were amplified using a polymerase chain reaction (PCR) method, and then the recombinant T7 phages containing the target genes were expressed on the phage surface. In addition, an indirect enzyme-linked immunosorbent assay (iELISA) method was adopted to screen the α' subunit epitopes which had been damaged by the heat treatments. Then, after three rounds of the expression and screening processes, the C1-Y fragments (488PHFNSKAIVVLVINEGEANIELVG511) were identified as α' subunit damaged epitopes. In order to confirm the types of epitopes, three peptides of the C1-Y fragments were synthesized using a solid-phase peptide synthesis method. The results of the iELISA showed that there was a linear epitope of 488PHFNSKAIVVLV499 in the C1-Y fragments. The results obtained in this study confirmed that the C1-Y fragments contained both linear and conformational epitopes, and that the conformational epitopes could be destroyed by thermal treatments. However, it was found that the linear epitopes could not be destroyed. The phage display protein Pt-C1-Y, coupling peptide BSA-Pep-Y, BSA-Pep-1, BSA-Pep-2, and soybean allergy patients' serum reaction results showed that only the Pt-C1-Y had reacted positively with the soybean allergy patients' serum. These findings further indicated that heat treatments can reduce not only the antigenicity, but also the allergenicity, of α' subunits in β-conglycinin.