| Food allergy is a front-burner issue of food safety,which has obtained widespread attention globally,shellfish is one of the eight major sources of food allergens proposed by the Food and Agriculture Organization.Scylla paramamosain is an important fishery resource in China,while its increasing consumption companied by an increasing in food allergy,thus a systematic analysis of allergens in S.paramamosain is crucial for the development of hypoallergic food and for the immunotherapy of shellfish allergy.In the present study,a total of 17,324 students was recruited in the epidemiological investigation on food allergy by distributing questionnaires in 2010-2017,and shell fish,mainly crab revealed the most allergic food among the validated questionnaires completed by 13,361 students.In a further serologic assay including 14,164 students from the total 17,324 students,4.31% of the tested sera showed positive reactivity to crab muscle,and among which 65.14% recognized crab myosinogen.Arginine kinase(AK)is a major allergen in S.paramamosain myosinogen,thus the crystal structure of AK was determined by X ray,and a structure with diffraction of 3.0 ? was achieved.A comparison of AK from S.paramamosain to AK of other species showed high sequence,secondary,and spatial structural identity.The similarity of the linear epitope regions among species,mainly distributed in amino acid(AA)25-44,AA130-155,and AA308-321,was observed in the epitope alignment of AKs;conformational epitopes were located in the structural conserved regions.Besides,in addition to triosephosphate isomerase(TIM)and sarcoplasmic calcium-binding protein(SCP),filamin C(FLN c)was identified as a novel allergen in S.paramamosain myosinogen for the first time.Results of serological assay showed that AK and FLN c can react with more patients' sera than TIM and SCP.These allergens were accepted by the world health organization/ international union of immunological societies(WHO/IUIS),and AK,SCP,TIM,and FLN c were named Scy p 2,Scy p 4,Scy p 8,and Scy p 9,respectively.For the confirming of anaphylaxis,subjects with crab allergy with clear clinical symptoms were identified by basophil activate test,the result of basophil activate test showed that the up-regulation of CD63 and CD203 c on the basophil was in accordance with the severity of self-report allergic symptoms from patients.IgE from crab allergic patients were recruited as a target protein for the biopanning of phage display random peptide library.In addition to 6 conformational epitopes of SCP,5 linear epitopes,and 8 conformational epitopes of AK were identified.Four linear epitopes and 2 conformational epitopes of TIM,6 linear and 6 conformational epitopes of FLN c were also identified.The purified allergens were used as antigens to build an in vivo allergy model in Balb/c mouse.AK showed the strongest allergenicity,with the ability of promoting B cell proliferation and the production of high level of antigen-specific IgE;TIM could also promote the proliferation of B cell and produce certain degree of specific IgE,while its allergenicity was relatively weaker compared to AK;SCP showed strong immunogenicity while weak immunoreactivity;as to FLN c,the immunogenicity was weak but its immunoreactivity was strong,which is able to induce strong activation of effector cells.In conclusion,our present study revealed the importance of crab myosinogen in food allergy.Crystal structure of the major crab myosinogen allergen,AK,was determined;purification,serological assay,and the epitopes mapping of AK,TIM,FLN c,and SCP were completed.The four myosinogen allergens have been accepted by the WHO/IUIS.In vivo experiment was performed for the further comparison of allergenicity of the four myosinogen allergens.The achievements above may lay the foundation for the development of hypoallergic foods and the immunotherapy of shellfish allergy. |
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