RESUMEN
Glycinin is an important allergenic protein. A1a is the acidic chain of the G1 subunit in glycinin (G1A1a), and it has strong allergenicity. In this study, we used phage display technology to express the protein of G1A1a and its overlapping fragments and an indirect enzyme-linked immunosorbent assay (iELISA) to determine the antigenicity and allergenicity of the expressed protein. After three rounds of screening, it was determined that fragment A1a-2-B-I (151SLENQLDQMPRRFYLAGNQEQEFLKYQQEQG181) is the allergenic domain of G1A1a destroyed by thermal processing. In addition, three overlapping peptides were synthesized from fragments A1a-2-B-I, and a linear epitope was found in this domain through methods including dot blot and iELISA. Peptide 2 (157DQMPRRFYLANGNQE170) showed allergenicity, and after replacing it with alanine, it was found that amino acids D157, Q158, M159, and Y164 were the key amino acids that affected its antigenicity, while Q158, M159, R162, and N168 affected allergenicity.
Asunto(s)
Alérgenos , Globulinas , Calor , Proteínas de Soja , Alérgenos/inmunología , Alérgenos/química , Humanos , Globulinas/química , Globulinas/inmunología , Proteínas de Soja/química , Proteínas de Soja/inmunología , Secuencia de Aminoácidos , Hipersensibilidad a los Alimentos/inmunología , Epítopos/química , Epítopos/inmunología , Dominios Proteicos , Antígenos de Plantas/inmunología , Antígenos de Plantas/química , Antígenos de Plantas/genética , Glycine max/química , Glycine max/inmunología , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
Glycinin is an important allergen in soybeans. In this study, molecular cloning and recombinant phage construction were performed to explore the antigenic sites of the glycinin A3 subunit that were denatured during processing. Next, the A-1-a fragment was located as the denatured antigenic sites by indirect ELISA. The combined UHP heat treatment showed better denaturation of this subunit than the single heat treatment assay. In addition, identification of the synthetic peptide showed that the A-1-a fragment was an amino acid sequence containing a conformational and linear IgE site, in which the first synthetic peptide (P1) being both an antigenic and allergenic site. The results of alanine-scanning showed that the key amino acids affecting antigenicity and allergenicity of A3 subunit were S28, K29, E32, L35 and N13. Our results could provide the basis for further development of more efficient methods to reduce the allergenicity of soybeans.