Study On The IPEC-J2s Absorption Of β-conglycinin And Its Hydrolysate

In recent years, researchers have made a deep study on soybean anti-nutritional factors and found soybean antigen could destroy intestinal barrier of young animals which severely limited the utilization of soybean in the animal husbandry. In this study, β-conglycinin and its enzymatic peptides were used as the main allergens and the intestinal epithelial cells of piglets(IPEC-J2) were used as the research model to investigate the effects of different concentrations of β-conglycinin and its enzymatic peptides on IPEC-J2 s absorption regularity and transport methods of the allergens through the epithelial cells at different time. In addition, genistein and fructooligosaccharide were used to screen the optimal concentration of inhibitors. This study will provide a basis for further studying the sensitization mechanism of soybean allergens. Experiment Ι: Study on the soybean antigen protein absorption dynamics of IPEC-J2sIn this trial, IPEC-J2 s were seeded in traswell and treated with different concentrations of β-conglycinin or enzymatic peptides(0、0.25、0.5、1.0 mg/mL) for(0、2、4、8、12 h),β-conglycinin immunoreactivity in apical/ basolateral solutions of transwell and cytoplasmic proteins at different times were further detected using ELISA and Western blot. Then corresponding stripes were separated by SDS-PAGE and identified using Q-E mass spectrometry. The results indicated that the amounts of β-conglycinin and enzymatic peptides with immunoreaivity had a significant increase in a dose-time dependent manner P<0.05). But the amount of intracellular accumulation was significantly decreased P<0.05). These results suggested that β-Conglycinin and enzymatic peptides passed through the IPEC-J2 by endocytosis.In order to verify the pathway of β-conglycinin and enzymatic peptides across the cells, IPEC-J2 s were treated with 1.0 mg/mL of β-conglycinin or enzymatic peptides for 8 h. Clathrin on the membrane surface were detected using immunofluorescence. The results indicated that the visible fluorescences appeared on the membrane surface of IPEC-J2 s treated with β-conglycinin and enzymatic peptides, and the bound of cells was clear and appeared a net structure. This suggested that the endocytosis pathway of β-conglycinin and enzymatic peptides was clathrindepended transport in IPEC-J2 s. Experiment ΙΙ Study on the screening of inhibitors of soybean antigen protein.In this trial, IPEC-J2 s were seeded at transwell and treated with different concentrations of genistein(0、6、12、18、24 μg/mL) and fructooligosaccharide(0、3、6、9、12 μg/mL), which were diluted in 1.0 mg/mL β-conglycinin or enzymatic peptides for 8 h. β-conglycinin immunoreactivity in apical/basolateral solutions of transwell and cytoplasmic proteins at different times were further detected using ELISA and Western blot. The results indicated that inhibitory effects of β-conglycinin groups were significantly lower than the enzymatic peptides groups when treated with same inhibitors P<0.05). However, the inhibition effects of genistein group were significantly lower than that in fructooligosaccharide group when treated with same protein as allergen(P<0.05). This suggested that fructooligosaccharide inhibited the absorption of the enzymatic peptides in IPEC-J2 s.In conclusion, with the increasing concentrations and treatment times of β-conglycinin and enzymatic peptides, their absorptions through clathrin-depended endocytosis were significantly increased in IPEC-J2 s. On this basis, adding appropriate concentration of genistein and fructooligosaccharide could effectively inhibit the absorption of soybean protein antigen. In sum, this research will provide a basis for further studying the allergic mechanism of soybean allergens and improving the use efficiency of soybean protein in animal nutrition.