Allergenicity Analysis Of The Cross-linked Tropomyosin Of Scylla Paramamosain

Recently,the consumption of seafood products is growing rapidly because of the improvement of people's living standard.It is crucial to figure out that crabs,as a kind of nutritious and delicious high quality protein popularizing among individuals,have caused numerous cases of food allergy which significantly influence human being's health condition.Although some of researches on eliminating allergens are conducted overseas,limited attention is paid regarding to this subject in China.How to reduce the allergenicity of allergen has become the research hot spot.Therefore,this paper tries to evaluate the impact of enzymatic cross-linked TM,and attempts to determine the relationship between crosslinking sites and antigen epitopes.It hopes that it can provide theoretical guidance on manufacturing production of hypoallergic crab and offers help on the treatment for other crustacean allergens.In this study,tropomysin(TM)was purified from Scylla paramamosain.The application of phage display technology,got nine TM conformation epitopes,its conformation epitopes is constituted by the two chain multiple amino acid,the conformational epitope of some amino acids is the same to linear epitope have been reported.Three enzymes will be applied in the crosslinking reaction.The tyrosinase and horseradish peroxidase can effectively promote the crosslinking and reduce the lgG/lgE-binding activity.The optimal conditions for the cross-linking of TM were as follows: horseradish peroxidase(300 U/?L,product was named CHP),or tyrosinase(450 U/mL,product was named CTC)reacted with TM(0.6 mg/mL)at 37? for 3 h.In the case of CTC,0.25 mM caffeic acid was added as a mediator.Furthermore,the allergenicity,the cross-linking degree,the change of secondary structure,the surface hydrophobicity,the digestion stability and the allergenicity were examined by proteomics and immunomics.In addition,the potential of sensizition and oral tolerance in vivo were analyzed by a TM-induced food allergy BALB/c mouse model.ELISA analysis revealed that the IgG-binding activity of CTC and CHP decreased by 16.9 ± 0.8% and 70.8 ± 0.5%,respectively and the IgE-binding activity of CTC and CHP reduced by 34.5 ± 1.8% and 63.5 ± 0.6%,respectively.The result of SDS-PAGE and SEM indicated that CTC and CHP were extensively cross-linked accompanying with the dominant higher molecular proteins.Due to the effect of enzymatic cross-linked reaction,the ?-helix percentage of cross-linked TM has reduced.Besides,enzymatic cross-linking reaction also can influence allergen of hydrophobicity,which the hydrophilic amino acids of TM were covered by the cross-linking of tyrosinase,but the hydrophilic amino acids of TM were exposed by that of horseradish peroxidase and then CHP increased digestibility by pepsin.Cell assay also reveals that CTC and CHP can considerably decrease the production of histamine and the release of ?-hexosaminidase and also affected the phagocytosis of DC cells.The crosslinked products can induce oral tolerance and reduce allergenicity in mice by decreasing IgE and IgG1 levels in serum,the production of T-cell cytokines,and the percentage composition of dendritic cells.These findings demonstrate CHP has more potential of reducing the allergenicity than CTC via influencing the morphology of protein,changing the original method of antigen-presenting,modulating the Th1/Th2 immunobalance,and induce the oral tolerance of the allergen tropomyosin.In addition,the conformational epitope of TM has been studied by using phage display technology and the paper also analyse the relationship between cross linking sites and antigen epitopes.The results demonstrated that enzymatic processing of TM by tyrosinase or HRP had a potential in reducing IgE binding and allergenicity.It might provide some new thought and methods for developing hypoallergenic aquatic food through enzymatic cross-linking.