Study On Distribution And Identification Of Soybean β-conglycinin And Its Major Epitopes With Piglet Intestinal Epithelial-binding Proteins

β-conglycinin is an important antigenic protein that causes intestinal sensitization in piglets.Soybean antigen epitopes in the digestive tract are the fundamental substance responsible for animal allergies.The digestive tract is the primary contacted tissue when food/feed allergen enters body.If its receptors occurre in the intestinal epithelium which could bind to it,it is crucially related to sensitization of piglets.But little study has been developed on the allergen’s receptors.To further explore the receptor,the identification of β-conglycinin and piglet intestinal epithelial binding proteins is particularly important.Therefore,this study compared the binding capacity of the soybean β-conglycinin(including its antigenic epitopes)and soybean allergic serum in different species,and then the antigenic epitopes with strong binding ability to piglet allergic serum was selected.Immunohistochemistry and co-immunoprecipitation were used to characterize the binding proteins of the soybean β-conglycinin(including its antigenic epitopes)and the intestinal epithelium in piglets.This study provides the scientific basis for further revealing the mechanism of soybean allergy and optimizing animal feed.Experiment I: Comparison of binding ability of β-conglycinin epitope with sensitized serum of piglets,calves and rats.In this study,ELISA and Dot-blotting were used to compare the differences in binding ability of β-conglycinin α subunit and β subunit with sensitized serum of three different species of animals.The results showed that there was a significant difference in the linear epitope-sensitive region where the soybean antigen protein β-conglycinin specifically binds to allergic sera of three animals,and the seven epitopes have a significantly higher binding capacity to allergic serum of sensitized piglets than rats and calves.At the same time,according to the test results,three peptides with strong binding ability to sensitized serum of piglets were selected as α2,β2 and β3,which provided the experimental basis for identifying the binding protein of piglet intestinal epithelium and β-conglycinin epitopes(Experiment II).Experiment II: Identification of β-conglycinin,its Epitopes,and intestinal epithelial-binding protein in piglets.Immunohistochemistry,co-immunoprecipitation and mass spectrometry techniques were used to identify soybean antigen protein(including its major epitopes selected according to the results of Experiment I)and piglet intestinal epithelial binding protein from the binding site and the type and function of the binding protein.The results of the histology showed that there were differences in the binding protein of β-conglycinin and its antigenic epitopes in different parts of the small intestine of piglets.Further experiments found that β-conglycinin and its antigenic epitopes and intestinal epithelial binding proteins of piglets can be divided into three categories: skeleton proteins,catalytic enzyme proteins,and functional regulatory proteins,and there are differences in the different epitopes and intestinal epithelial binding protein types of piglets.The research results provide an important basis for revealing the causes of sensitization of piglets and lay a foundation for further analysis of soybean antigen receptors in intestinal epithelial cells.