Effect of Simulated Intestinal Fluid on Immunogenicity of Pen a1 and Its Epitopes
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Abstract:
Simulated intestinal fluid (SIF) was used to digest Pen a1, and changes in the immunogenicity of Pen a1 after digestion were analyzed. After digestion of shrimp antigen Pen a1 and the epitope peptides of Pen a1, a Pen al antibody and epitope-specific antibody were used to measure changes in the immunogenicities of Pen a1 and its epitopes, and the digestive stabilities of the epitope peptides of Pen a1 were determined. The results showed that the immunogenicity of Pen a1 decreased significantly within 60 min of digestion and continued to decrease slowly after 90 min. The generated protein fragments remained immunogenic, but were gradually degraded until they could no longer be detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Similar changes were observed for the immunogenicities of five epitopes of Pen a1. Western blot results indicated that the binding degrees of the five epitope antibodies with the proteolytic fragments of Pen a1 after the SIF digestion were different. The numbers of 20~30 ku fragments bound to antibody No. 3, 4, and 5 were greater than those bound to antibody No. 1 and 2. Enzyme-linked immunosorbent assay results indicated that after 4-h digestion, the immunogenicity had only decreased by 80%. The digestive stabilities of the five epitopes of Pen a1 were as follows: No. 2 > No. 1 > No. 3 > No. 4 > No. 5. In contrast, the digestive stabilities of the five epitope peptides of Pen a1 were in the following order: No. 3 > No. 1 > No. 4 > No. 2 > No. 5, which was negatively correlated with the number of cleavage sites on the epitope peptide. In conclusion, epitope No. 2 showed the highest digestive stability, while epitope No. 5 showed the lowest digestive stability.
