Allergenicity Analysis Of Enzymolysis On Shrimp Allergenic Protein

Shrimp is one of the most popular consumption seafood, may cause a serious allergic reaction.Every year there is a large number of people getting allergy due to eat food which include shrimp. It constituted a serious danger to people’s healthy, and a great waste of food resources. So it’s very necessary to make a research on the allergenicity of shrimp. With the further study of shrimp allergens, there is a large number of reports had appeared, however, such reports were mostly using ELISA to indicated allergenicity; and repots about enzymolysis make the change to antigen spatial structure which lead to allergenicity changes were rarely. In this study, Shrimp protein was extracted by PBS, Pen a 1 was purified by salting-out and isoelectric precipitation, and prepared polyclonal antibody with the Pen a 1. The impact of enzymolysis on shrimp allergenic protein was analyzed by CD spectroscopy and polyclonal antibodies. At last a BALB/c mouse shrimp allergenic model was established to evaluation allergenicity of protein hydrolysates. Conclusion: The enzymolysis on shrimp allergenic protein can reduce the antigenicity, thus also reduced the Allergenicity to a certain extent. But after several immune processes, protein hydrolysates were still has very strong allergenicity. The research contents are changes of the shrimp allergenic protein antigenicity and allergenicity impact on a mouse model; we are now laying the groundwork for the allergenicity reduction mechanism of allergenic protein.According to Pen a 1 related characteristics, using a salt solution extraction and purified by some methods like salting-out, Prepared the Pen a 1 successfully. And the preparation conditions were determined: Extract by PBS 1:10(W/V); 35% sammonium sulfate salting out; Isoelectric point precipitation p H4.5; Removed the impurity proteins by heating 15 min. After identification, the protein was identified as shrimp allergenic protein, Pen a 1 obtained by the method also have good activity.Pen a 1 mixed with freund’s adjuvant, immune the New Zealand rabbit,Serum with Protein A purification, the Antibody titer greater than 80000,IC50 has been calculated from the inhibition curve, the value is 0.0448μg/m L. The results showed that the antibody is good.The enzymolysis results of various enzymes showed that the alkaline protease hydrolyzes shrimp allergenic protein best. The optimal condition of alkaline protease was optimized by single factor and orthogonal test. The optimized conditions are as follows: p H9;Temperature 50 ℃;Enzymolysis time is 3h;Enzyme substrate is 3000 μ/g. The results of CD spectra showed that: The enzymolysis made the α-helix ratio of shrimp allergenic protein decreased by 3.5%, the β-sheet ratio decreased by 1.5%, the β-turn and Random ratio were increased by 4.3% and 1.7%.A BALB/c mouse shrimp allergenic model was established with shrimp allergenic protein and its hydrolysates. The s Ig G, s Ig E and histamine in serum were measured by ELISA, the spleen indexes and allergic symptoms of mice were observed. The results showed that: The s Ig E of hydrolysates was reduced by 40.44%. But the results of s Ig G and spleen index showed that the shrimp allergenic protein group and protein hydrolysates group had no significant difference. And the protein hydrolysates group paraffin sections of intestine showed that the inflammatory reaction more obvious than the shrimp allergenic protein group.