Effects On Immunogenicity Of Pen A1 And Its Epitopes By Simulated Human Digestion

Food allergy has become one of the serious food safety problems with the development of food processing industry. Shrimp is an important allergenic food in China, and lots of people have suffered shrimp allergic reactions. The studies on food allergy showed that heating, irradiation, high pressure, enzyme hydrolysis and so on could reduce the sensitization of shrimp allergencity. The structure, epitope, amino acids and other key information of shrimp allgern Pen a1 are alraedy investigated and identified. However, human digestive system would destructure the whole protein and its epitope of the allergen, but the digestion effect on the sensitization of allergen was not clear. This study will focus on the immunogenicity of allergen and its epitopes during human digestion, which has important theoretical significance on food allergy mechanism research and the preparation of desensitization food.In our experiment, the used serum were prepared by immuning New Zealand rabbits with purified Pen a1 and five synthesized epitope peptides No.1-No.5(Epitope1:Pen a143-57; Epitope2:Pen a185-105; Epitope3:Pen a1133-148; Epitope4:Pen a1187-202; Epitope5:Pen a1247-284). In order to evaluate the digestion stability of Pen a1, the immunogenicity of Pen a1 and its epitopes digested by simulated gastric, intestinal, and gastrointestinal fluid were detected by antibodies of Pen a1 and its epitopes. The specific contents and results are listed as follows:(1) Pen a1 was extracted and purified by KCl, ammonium sulfate fractionation and isoelectric precipitation. The epitope peptides were synthesized by using Fmoc method. The New Zealand rabbits were immunized with Pen a1 and five epitope peptides to obtain the whole protein antibody and epitope antibodies. These antibodies were tested and showed high sensitivity and specificity.(2) The results of simulated gastric fluid(SGF) digestion within 240 min: the immunogenicity of Pen a1 and its epitopes decreased signif icantly with the increasing of digestion time. The digestion stability of the whole Pen a1 protein and the epitope peptides varied. The stability of Pen a1’s epitopes was No.4 > No.2 > No.1 > No.3 > No.5; the stability of epitope peptides was No.1 > No.2 > No.3 > No.4 > No.5. Combined with the results of Western-blot, No.4 epitope of Pen a1 had the highest stability in SGF. Stability of No.1, 2, 3 was lower than No.4, and there were almost no signif icant differences among them; No.5 epitope showed rather rapid degradation in SGF than others.(3) The results of simulated intestinal fluid(SIF) digestion in 240 min: the immunogenicity of Pen a1 and its epitopes decreased fast within 60 minutes and then decreased slowly after 90 minutes. Some new protein fragments which generated during digestion also showed allergenic and the fragments were not detectable till 180 min digestion. Ci-ELISA also showed the difference between the SIF digestion stability of Pen a1’s epitopes and epitope peptides: the epitopes of Pen a1 was No.2 > No.1 > No.3 > No.4 > No.5; the stability for epitope peptides was No.3 > No.1> No.4 > No.2 > No.5. There was a negative correlation between the digestion stability and the quantity of cleavage sites of epitope peptide. It is concluded that No.2 epitope of Pen a1 had the highest stability, and No.5 epitope was the most labile to SIF digestion.(4) The results of simulated gastrointestinal fluid(GI) digestion(120 min SGF digestion and 240 min SIF digestion): Pen a1 degraded into many fragments with the increased digestion time and these proteolytic fragments inordinately bound to six antibodies tested by SDS-PAGE and Western-blot. The whole antibody bound to almost all proteolytic fragments, antibodies of No.3, 4 were lower than whole antibody, No.5 was follewed, and No.1, 2 had almost no reaction to these small fragments. T he capacity of IgE-binding of Pen a1 and its epitop e peptides by GI digestion was analyzed quantitatively by means of Ci-ELIS A. The result showed that the digestion stability of the epitopes of Pen a1 was No.2 > No.4 > No.3 > No.1 > No.5, and the epitope peptides’ stability was No.3 > No.1 > No.2 > No.4 > No.5. So No.2 epitope had the highest stability, and No.5 epitope showed rather rapid degradation in GI than others.The result of the above three digestion conditions showed that the digestion stability of No.4 epitope was the highest in SGF but was poor in SIF, and showed the relatively low level of stability after continuously gastrointestinal(GI) digestion. No.2 epitope had the highest digestion stability in GI and also showed high stability both in SGF and SIF because of the protection of the spatial structure of Pen a1. No.5 located in the end of Pen a1 and had long binding region and more cleavage sites, which made it the lowest stability in all the three digestion conditions.It is concluded that No.4 epitope had the highest digestion stability in SGF; No.2 epitope had the highest digestion stability both in SIF and GI; No.5 epitope was the most labile epitope to all the three digestions. The results of this study could be used in the treatment of allergenic patients and in the future research of allergy mechanism.