Immunoreactivity Variation Of Epitopes In Shrimp Allergen Under Irradiation And Heat Treatment And Analysis On Amino Acids Of Epitopes

Seafood and its product have been enjoyed by people for a long time for its delicacy and nutritional value. But shrimp, crab and fish also belong to the eight kinds of allergenic food according to FAO statistics. In recent years, food allergen by seafood are more and more serious with the consumption on seafood becomes more popular, which make a great threat on people’s health, and caused attention of people from all over the world. More and more scholars devote themselves to the ways of desensitization and the allergenic mechanism. So far, the desensitization way studied most are physical ways like heat and irradiation. But studies of processing methods on desensitization are direct on macroscopic aspect, studies on the influence on epitopes are lesser. And the studies on the sequence of amio acids in epitopes have important theoretical significance on desensitization and reduced-allergy food production.In our experiment, Pen al was extracted and purified from shrimp, and then under heat, irradiation and heat combined with irradiation treatment. The Pen al with Pen al serum and five epitope sera were used to analyze the influence of different processing ways on the sensitization of Pen al, and the imunoreacticity variation of five epitopes under different treatment. Study the crutial amino acids of epitopes using Ala mutation. The specific research contents are as follows:(1) Pen al extration and purification and the polyclonal antibody preparation. Extract the Pen al with KCl, purified with isoelectric precipitation, ammonium sulfate precipitation and gel chromatography. Use Pen al as immunogen to immune Newzeland rabbits, the tilter of Pen al antiserum reached to8.92×108.(2) Pen al under treatment of heat and irradiation, detect the variation of immunoreactiviy by western blot and Ci-ELISA. Heat Pen al for different time (0,5,15,30min), treat Pen al with y-irradiation (0,7, lOkGy), heat and irradiation treatment (5min,7kGy,12kinds of ways). The western-blot and Ci-ELISA results showed that heat alone cannot decrease the allergenicity of Pen al and the five epitopes, on the contrary, allergenicity increased with the extension of time. Irradiation with7kGy cannot decrease the allergenicity, irradiation with10kGy can decrease the immunoreactivity of Pen al. When under irradiation and heat treatments, it has good effect on desensitization. The analysis on the immonoreactivity of five epitopes showed that epitopel,2,4were sensitive to irradiation and heat treatment, and the allergenicity increased. The variation of epitope3was consistent with Pen al, the allergenicity decreased after irradiation,and when combined with heat, the effect was better. The IC50value of epitope5hardly changed, meant that epitope5was stable to irradiation and heat treatment, could be used in immunoassay kits.(3) Design and synthesis of epitope peptides mutation and measure of the allergenicity. Analyzed the amino acids’ frequency of occurrence and their conservative property, synthesized the mutants, and analyzed the IgE capacity of the mutants, screened out the critical amino acids of the epitope of Pen al. Substituted them to Ala, and synthesized the mutants with Fmoc method. The mutants were interacted with epitope sera to detect the IgE reactivity by competitive Dot-blot and ELISA. Results showed that the crutial amino acid of epitope1was Gln, the crutial amino acids of epitope2was Leu and Glu, the crutial amino acids of epitope3was Leu and Asp, the crutial amino acids of epitope4was Val,Leu and Glu, the crutial amino acids of epitope5was Leu.In conclusion, we analyzed the effect of different treatment such as heat and irradiation on the allergenicity of Pen al, and studied the variation of the five epitopes under the treatment to analyze the function of epitopes on the allergenicity of Pen al. Besides, by analyzing the amino acids of five epitopes, Ala mutation, we selected the crutial amino acids of each epitopes, which offered theoretical basis for explitation of reduced-allergy food and reaseach of allergenic mechanism.