Absorption And Transport Mechanism Of Soybean β-conglycinin In Small Intestinal Mucosa Of Piglets

Soybean has high nutritional value and is rich in protein and amino acids,and known as "the king of beans".Therefore,it is widely used in feed as a source of plant protein.However,the antigen protein in soybean can induce anaphylaxis and diarrhea in weaned piglets,which affect the performance of piglets and bring losses to the pig industry.Soybean β-conglycinin is the most immunogenic antigen protein.In this study,different levels of β-conglycinin were used to replace non-soybean protein sources in the diets,and the absorption and transport mechanism of macromolecular antigen peptides from β-conglycinin in piglets’ intestines was explored in order to provide theoretical basis for preventing and treating diarrhea caused by soybean antigen protein,improving piglet’s health and increasing feed utilization rate.(1)Separation and purification of β-conglycinin and preparation of rabbit polyclonal antibody against β-conglycininIn this experiment,β-conglycinin was separated according to the isoelectric point property,and then purified by salting out method.The purified β-conglycinin was used to immunize New Zealand white rabbits,and the titer of the prepared anti-β-conglycinin serum was 1:3280500.(2)Effects of β-conglycinin on growth performance and immune indexes of weaned pigletsEighteen 10-day-old piglets were randomly divided into three groups,and the feeding trial lasted 14 days.The piglets in control group were fed a basic diet without soybean protein source.While the piglets in the low group and high group were fed the trial diets formulated by replacing casein in basal diet with 2% and 4% β-conglycinin,respectively.After the experiment,piglets were slaughtered and sampled.The results showed that the daily feed intake and daily gain of piglets in β-conglycinin groups decreased significantly(P <0.01)compared with the control group.The diarrhea incidence of piglets enhanced with the increase of the supplementation amount of β-conglycinin in diets.With the increase of dietaryβ-conglycinin content,the levels of total Ig E,specific Ig E in serum and IL-4 in spleen cell culture solution increased significantly(P<0.05),while the level of IFN-γ decreased significantly(P < 0.05)compared with the control.In 4% β-conglycinin group,the height of jejunal villi decreased significantly(P<0.01)and the depth of crypt increased significantly(P<0.05),and the ratio of villi to crypt decreased significantly(P < 0.05)in comparison with the control.The content of VFA in piglet’s cecum had an increased tendency with the increase of β-conglycinin content in diet.The butyric acid content in the high group,however,was significantly higher than that in the control group and the low group(P<0.05).(3)Absorption and transport mechanism of β-conglycinin in intestinal mucosa of pigletsIn this experiment,Western blot and polypeptide sequencing were used to identify the antigen fragment types of β-conglycinin digested and absorbed in small intestine of piglets.Immunofluorescence and immunoelectron microscopy were used to locate the antigen molecules of β-conglycinin in jejunum intestinal mucosa of piglets,and comparative proteomics and transcriptomics were used to screen the differential proteins and genes between the high group(4% supplementation group)and the control group.The results showed that the macromolecular antigen peptide of β-conglycinin were absorbed and transported into the intestinal mucosa in the form of Ig E-antigen complex with CD23 as carrier through transcellular pathway.The α,α’ and β subunits of β-conglycinin and their52-70 k Da antigenic peptides were absorbed into the intestinal mucosa and many small molecular antigenic peptides(< 10 k Da)were absorbed into the blood circulation.There were292 differentially expressed protein in jejunum between 4% β-conglycinin group(high group)and control group,which mainly represented immune response and transcellular transport.Howvere,the function of differentially expressed genes in jejunum between 4% β-conglycinin group(high group)and control group was mainly reflected in some biological processes.