大豆抗原蛋白β-conglycinin在仔猪小肠粘膜吸收转运机理的研究

Post-weaning diarrhea in piglets is a difficult problem that has been puzzling the pig industry. The allergic reaction induced by soybean antigens such as β-conglycinin is the primary factor for the post-weaning diarrhea in piglets in modern pig production which is based on the corn and soybean meal-based diet. It is a key step to inducing allergic reaction in piglets that β-conglycinin get into body throught the small intestinal mucosa in the form of large antigen polypeptide fragments. However, the polypeptide fragments of β-conglycinin that is absorbed and transported in the small intestinal mucosa of piglets remain to be identified, and the pathway and molecular mechanism by which β-conglycinin is absorbed also need to be clarified. The antigen polypeptide fragments that are absorbed and transported in the small intestinal mucosa will be identified by the 2D-western blot & mass spectrometry using the piglets as experimental animal. Laser scanning confocal microscopy, flow cytometry, RNAi, qPCR, WB and immuno- fluorescence cytochemistry will be utilized to explore the pathways and means, and the molecular mechanism by which these polypeptide fragments are absorbed and transported by in vivo and in vitro experiments.The present work will reveal the absorption and transport mechanisms of β-conglycinin in the small intestinal mucosa of piglets at the cellular and molecular level, and give new ideas for the prevention and treatment of post-weaning diarrhea in piglets.

仔猪断奶腹泻一直是困扰养猪业的难题，在以玉米-豆粕型日粮为基础的现代养猪生产中，β-conglycinin等大豆抗原蛋白引起的肠道过敏反应是仔猪断奶腹泻的原发性因素。β-conglycinin以大分子抗原肽段的形式经小肠粘膜进入机体是其诱发肠道过敏反应的关键环节，但β-conglycinin在仔猪小肠粘膜吸收转运的肽段种类、吸收转运的途径和方式以及分子机制尚不清楚。本项目拟以仔猪为试验动物，通过在体和离体试验，利用2D-WB结合质谱鉴定β-conglycinin在仔猪小肠粘膜吸收转运的抗原肽段种类；分别利用激光扫描共聚焦显微镜技术、流式细胞术、RNAi技术，以及qPCR、WB和免疫荧光细胞化学技术等探讨这些肽段吸收转运的途径和方式及分子机制，从而在细胞和分子水平上阐明β-conglycinin在仔猪小肠粘膜的吸收转运机理。本项目将为防治仔猪断奶腹泻提供新思路。

断奶是仔猪培育的关键环节，仔猪断奶尤其是早期断奶容易引起腹泻，这一问题一直是困扰养猪业的难题。仔猪断奶腹泻发生机制复杂，影响因素众多，但以玉米-豆粕型日粮为基础的现代养猪生产中，大豆抗原蛋白引起的肠道过敏反应是仔猪断奶腹泻的原发性因素。大豆抗原蛋白以抗原肽段的形式经小肠粘膜进入机体是其诱发肠道过敏反应的关键步骤。但β-conglycinin等大豆抗原蛋白在仔猪小肠粘膜吸收转运的抗原肽段种类、吸收转运的途径和方式以及分子机制尚不清楚。本项目以早期断奶仔猪为试验动物，通过饲喂含大豆抗原蛋白日粮建立仔猪过敏模型，利用WB和质谱技术检测胃肠道食糜中抗消化抗原肽段（肽）及吸收进入小肠粘膜和血液循环的抗原肽段（肽），明确了大豆抗原蛋白β-conglycinin在仔猪小肠粘膜吸收转运的多种可吸收肽段（肽）；利用体内试验通过免疫荧光和免疫电镜技术证明大豆抗原蛋白β-conglycinin通过跨细胞途径进入机体，体外细胞试验进一步发现大豆抗原蛋白在仔猪小肠上皮细胞的吸收动力学符合米氏方程，并通过网格蛋白介导、小窝蛋白介导的内吞作用进入上皮细胞实现跨细胞转运；在此基础上，进一步通过体内试验探究了大豆抗原蛋白对小肠粘膜IgE低亲和力受体（FcεRII）和IgE的影响以及FcεRII、IgE和抗原肽段（肽）的共定位情况，结果表明，随着仔猪日粮中大豆抗原蛋白β-conglycinin添加量的增加，仔猪小肠粘膜FcεRII、IgE水平显著升高，同时在仔猪小肠粘膜中存在FcεRII、IgE和抗原肽段（肽）的共定位。通过以上研究可以初步得出结论：仔猪断奶后采食含大豆抗原蛋白的日粮，大豆抗原蛋白的多种抗原肽段（肽）被吸收进入小肠粘膜组织和血液循环，引发免疫应答和过敏反应。大豆抗原蛋白β-conglycinin主要通过跨细胞途径借助IgE低亲和力受体以抗原肽段（肽）-IgE抗体复合物的形式被仔猪小肠粘膜上皮细胞吸收和转运。本项目的研究结果可为预防和治疗大豆抗原蛋白引起的仔猪肠道过敏和腹泻提供新思路，并为寻找新的药物靶点和开发有效治疗药物提供理论依据。