| Food allergy is a front-burner issue of foodsafety which has obtained widespread attention globally, crustancea is one of the eight major sources of food allergens proposed by the Food and Agriculture Organization(FAO). Crab is an important fishery resource for its high nutritional value and delicious taste. With the consumption of crab increasing, reports of allergy diseases induced by crab are growing. It was reported that Arginine kinase(AK) is a panallergy present in crustaceans, which can induce an immunoglobulin(Ig) E-mediated immune response in humans, as a consequence, a full picture of epitopes of AK is essential if we are to develop hypoallergic food or to establish new and better diagnostic and therapeutic tools for food allergies.In the present study, AK was purified from Scylla paramamosain, and then digested by simulated gastric fluid(SGF) in vitro, the result of immumoblotting indicated that the pepsin hydrolysed AK has Ig G/Ig E binding activity. The pepsin hydrolysate of AK were separated and purified by high performance liquid chromatography(HPLC), and five Ig G/Ig E actived peptides wre obtaind. Native AK and partial recombinant AKs(r AK1: 87-186, r AK2: 172-265) were used as antigens to build AK-induced food allergy mouse model. The level of specific Ig E, IL-4 and IL-13 of native AK and r AK1 groups showed a significicant increase after second intraperitoneal injection, indicating that mice from native AK and r AK1 had suffered from allergy, and epitopes of AK may mainly distributed in the region of r AK1.Specific rabbit antibody was raised against S. paramamosain AK from New Zealand white rabbits immunized. Rabbit-anti-AK Ig G was purified and used for immunoscreening of a phage displayed random peptide library, and as a result, five AK mimotope clones were obtained. Based on the biopanning result, four conformational epitopes C-AK-1(D3A4K43M1A5T49T44I7), C-AK-2(L31K33V35T32E11E18F14S34D37), C-AK-3(V177G172M173D176Q178T174L181K175L187), and C-AK-4(R202L170Y203E190P205W204L187T206Y145) were identified with the program Loca Pep, and mapped to S. paramamosain AK. The key amino acids of these conformational epitopes were D3, K33, T174, and W204, respectively.On the basis of biopanning, 19 peptides of AK were designed and synthesized, among which seven Ig G-specific peptides(AA: 113-127, 127-141, 134-148, 141-155, 204-218, 211-225, and 316-330) and six Ig E-specific peptides(AA: 113-127, 127-141, 141-155, 204-218, 211-225, and 316-330) were confirmed using the sera from crab-allergic patients, and four seropositive peptides(amino acids 113-127, 127-141, 141-155, and 204-218) were identified as linear epitopes in a degranulation assay in RBL-2H3 cells and named as L-AK-1, L-AK-2, L-AK-3, and L-AK-4, respectively.Stability experiments were performed to investigate the structure-activity relationship of AK. Native AK was denatured by SDS, β-Me, DTT, urea, and Gdn HCl, respectively, and then the Ig E-binding activity of denatured AK was detected by dot blotting, the results showed that the structural integrity of AK is essential for its allergenicity, and the intramolecular disulfide bond at Cys201-Cys271 is essential for its structural stability.In conclusion, in the present study the conformational and linear epitopes of S. paramanosain AK have been mapped, the sensitization relative bond and its structure-activity relationship has also been clarifie. These achievements may lay the foundation for developing hypoallergic foods and protection or therapy of allergic diseases. |
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