Purification, Identification And Characterization Of Allergen Parvalbumin In Freshwater Fish

Fish become a favorite food because of its rich protein and fatty acids. But fish allergens are also an important source of threat to human health. Studies have shown that major allergen of freshwater fish is parvalbumin. So research of parvalbumin allergen is particularly important.In this study, muscles of several freshwater Cypriniformes fishes were regarded as raw materials, a method for rapid purification of parvalbumin was established. Heating, two runs of trichloroacetic acid(TCA)precipitation were used to purify parvalbumin at first. Boiling could remove most of other proteins. Parvalbumin enriched a lot after twice TCA precipitation, there was only one other protein band by analysis of SDS-PAGE. By sephacryl S-200 HR acrylamide dextran gel filtration chromatography, parvalbumin of high purity(>90%) in freshwater fish was obtained. Because of the parvalbumin of high purity have characteristic absorption peaks in UV spectrum, UV absorption peak of purified parvalbumin in freshwater fish showed that the purified protein have characteristic absorption peaks during 200nm-350 nm in this experiment, it proved that the purified protein is parvalbumin of high purity. Further biological information of the purified protein was obtained by MALDI-TOF–TOF MS.In this experiment, parvalbumin monoclonal antibodies EG8 hybridoma were used to produce parvalbumin monoclonal antibodies. Parvalbumin monoclonal antibodies EG8 hybridoma were injected to BALB/C mice to prepare ascites. Parvalbumin monoclonal antibodies EG8 of high titer(≥80000) and good specificity were separated and purified. Results of SDS-PAGE and western blotting of purified parvalbumin monoclonal antibodies showed that the purity of parvalbumin monoclonal antibodies was high.Next, competitive inhibition ELISA and western blotting was processed to study the immunological cross-reactivity of purified parvalbumin of carp and crude extract protein of Hypophthalmichehys molitrix(silver carp), Ctenopharyngodon idellus(grass carp), Carassius auratus(crucian carp) in this experiment. Results of western blotting showed that cross reactivity existed indeed, the reaction molecular is about 12 KDa. ELISA competitive inhibition curve was used to obtain cross-reactivity rate. Cross-reactivity rate of purified parvalbumin of carp and crude extract protein of grass carp, crucian carp,silver carp were 83.34%, 96.35%, 60.93% separately.The analysis of vitro simulated gastrointestinal fluid of purified parvalbumin in carp and silver carp revealed that parvalbumin were degraded in the role of pepsin with a high speed.Molecular weight of degradation bands is small. Degradation rate of silver carp was slower than carp. Under the action of chymotrypsin and trypsin, carp and silver carp parvalbumin had no apparent degradation. Further analysis results by ELISA inhibition of gastrointestinal digestion products of carp and silver carp parvalbumin showed that sensitization of parvalbumin digestion products decreased a lot with pepsin after 1h.When digestion product was diluted, the sensitization decreased. Allergenicity of digestion products of silver carp parvalbimin decresced less than carp parvalbumin. Results of EILISA showed that there were major damage on parvalbumin allergenic with pepsin, but digestion products still retained a certain amount of allergenicity. The parvalbumin of carp and silver carp both showed a great tolerance with trypsin and chymotrypsin, the sensitization only had a relatively small decline compared to control group. Study on PH stability of carp parvalbumin found that carp parvalbumin began to degrade in p H 2.0, but was relatively stable in alkaline solution. Parvalbumin had no obvious degradation after sonication.