关键词: Pen a 1, SGF, 抗原表位, 消化稳定性

Abstract: 【Objective】 Pen a 1 is a major allergen in shrimp, and its epitopes play a decisive role in allergic reactions. In order to evaluate the digestion stability of Pen a 1, the immunogenicity of Pen a 1 and its epitopes digested by simulated gastric fluid (SGF) was detected by antibodies of Pen a 1 and epitopes. It provided a theoretical basis for the study of the human digestive effects on Pen a 1 and the mechanism of desensitization food preparation.【Method】Pen a 1 was purified from Metapenaeus ensis. Five epitope peptides of Pen a 1 were synthesized using Fmoc Method. The peptides were conjugated to keyhole limpet (KLH) and bovine serum(BSA) to get artificial immunity and coating antigen, respectively. The tested serum was prepared by immuning New Zealand rabbits with Pen a 1 and the artificial immune. SGF was prepared according to the method of United States Pharmacopoeia. The changes of molecular weight of Pen a 1 was observed by SDS-PAGE and Tricine-SDS-PAGE. The capacity of IgE-binding of Pen a 1 and its epitope peptides by SGF digestion was analyzed by means of Western-blot qualitatively and ci-ELISA quantitatively.【Result】SDS-PAGE showed that Pen a 1 was degraded with increasing digestion time and generated a new 22 KD sensitization allergic protein. Tricine-SDS-PAGE showed that there were no protein fragments generated between 1.7-18 KD. Western-blot indicated that after the digestion of Pen a 1, the proteolytic fragments inordinately bound to six antibodies. Antibodies of Pen a 1 and No.4 bound to almost all proteolytic fragments. The fragment of 22 KD was resistant to digestion. This fragment bound to antibody No.3, 4, and 5 but almost no reaction to antibodies No.1, and 2, which indicated that the fragment carried No.3, 4, and 5 epitopes. The inhibition rate between Pen a 1 after SGF digestion and the antibodies tested by ci-ELISA was significantly decreased, indicating that the immunogenicity of Pen a 1 and its epitopes was decreased significantly. Moreover, the epitope peptides digested by SGF got the same result, but the digestion stability between epitopes of Pen a 1 and epitope peptides varied greatly: the epitopes of Pen a 1 was No.4＞No.2>No.1> No.3＞No.5, and there was no significant differences among No.3, 1, and 2; the epitope peptides was No.1> No.2> No.3＞No.4＞No.5, and there was no significant difference among No.2, 3, and 4. Combined with the results of Western-blot, No.4 epitope had the highest stability in SGF but the stability of No.4 peptide wasn’t higher than others significantly due to the protection of the spatial structure of Pen a 1. Stability of No.1, 2, and 3 was lower than No.4, and there was no significant differences between them. No.5 epitope showed rather rapid degradation in SGF than others.【Conclusion】The method for detection of digestibility of allergen and its epitopes using epitope antibodies was established. The immunogenicity of Pen a 1 after SGF digestion reduced significantly, but the generated fragments were still possible to cause hypersensitivity. No.4 epitope had the highest stability after SGF digestion, and No.5 epitope to SGF digestion was the most labile.

Key words: Pen a 1, SGF, epitopes, digestibility