Comparative Studies Of β-conglycinin And Its Enzymatic Peptides On Mechanical Barrier Function Destruction In IPEC-J2

Soy protein allergy can cause significant damage to livestock production. It could destroy intestinal barrier function of young animals( such as pigs), causing malabsorption and diarrhea. β-Conglycinin and its enzymatic peptides,the major antigen protein in soybean, are selected as the allergens.In this paper, we selected piglet intestinal epithelial cells(IPEC-J2) as in vitro research model. We mainly studied the effects of different concentrations of β-Conglycinin and its enzymatic peptides on the mechanical destruction of intestinal epithelial barrier function.These results provide an important theoretical basis for further exploration of intestinal destruction mechanisms induced by soybean. Experiment I: Isolation and identification of immunoreactive activity of soybean antigen protein β-Conglycinin and its enzymatic peptides.In this test,β-Conglycinin is hydrolyzed continuously by pepsin and trypsin.Enzymatic peptides were separated by Gel chromatography and ultrafiltration,then they were identified by SDS-PAGE electrophoresis and Q-E mass spectrometry.The results showed that : These three peptides have immuneactivity with the molecular weight of 52 kDa, 30 kDa, and 25 kDa, respectively. The first peak is the 52 kDa enzymatic peptide after gel chromatography separation and purification. Enzymatic peptides mixture of 30 kDa and 25 kDa were obtained after ultrafiltration. Experiment II: Different concentrations of soy protein antigen β-Conglycinin and its enzymatic peptides affect on IPEC-J2.In this test, IPEC-J2 were treated by different concentrations(0、0.125、0.25、0.5、1 or 2 mg/mL)of soy protein antigen β-Conglycinin and its enzymatic peptides.MTT,TEER and AP were measured after incubation with different hours(2 h、4 h、6 h、8 h、12 h or24 h),in order to detect the effect of β-Conglycinin and its enzymatic peptides on cell integrity.The results showed that there were significantly different effects on epithelial integrity among the treatment groups, the strongest damage to cells incubated by mixed enzymatic peptides of 25 and 30 kDa,followed by hydrolysis of the total product(P<0.05).The TEER and MTT value in β-Conglycinin or enzymatic peptides treatments decreased in a time- a dose-dependent manner(P <0.05), and AP activity was significantly increased(P <0.05) after 24 h incubating. Conclusion:β-Conglycinin and its enzymatic peptides destroyed the cell proliferation activity, resulting in a negative impact on IPEC-J2 barrier. Experiment III:β-Conglycinin and its enzymatic peptides affect on the impact of the distribution and relative expression levels of the intestinal epithelial tight junction proteins(ZO-1, Occludin, Claudin-3 and Claudin-4).To further study the related mechanisms of allergen on IPEC permeability changes, IPEC-J2 were treated by β-Conglycinin and its enzymatic peptides(0.5mg/mL) with 24 h, to detect distribution and relative expression of Tight junction(ZO-1、Occludin、Claudin-3 and Claudin-4)in cell. The results showed that the fluorescence staining of tight junction turned to blur and loose after allergens incuabting. Tight junction(ZO-1、 Occludin、 Claudin-3 and Claudin-4)protein relative expression were reduced compared with control group(P <0.05) by western blot analyses. Conclusion: allergens undermine the integrity of the tight junction protein, making its protective epithelial barrier dysfunction.In summary:β-Conglycinin and its enzyme hydrolyzed peptides can enhance its mechanical barrier function injury of IPEC-J2. The effect of enzymatic peptides damage to IPEC-J2 is higher than β-Conglycinin. In each enzymatic peptide, low molecular weight peptides destructive enzyme is the most serious, followed by hydrolysis of the total product, 52 kDa peptide hydrolysis is the lighter.Above results provide a reference for further reveal the mechanism of soybean allergens.