枯草芽孢杆菌诱导绵羊胃肠道防御素SBD-1表达的有效成分确定及其机理研究

抗生素引起耐药菌的出现和药物残留等问题对人、动物及环境造成的危害日益突出。我国从2020年起在饲料中全面“禁抗”。防御素是动物体内产生的一类内源性抗菌肽，益生菌不仅能够提高动物的饲料利用率，还能增加防御素的产生，是抗生素的理想替代品。而益生菌诱导防御素表达的有效成分及机理尚不完全清楚。本课题组已证明益生性枯草芽孢杆菌能诱导绵羊瘤胃上皮细胞内防御素SBD-1表达增多。本项目首先拟提取枯草芽孢杆菌细胞壁和培养上清及其成分；然后在体外培养的绵羊瘤胃和空肠上皮细胞内分别加入提取的不同成分共培养；再采用qPCR和ELISA方法，从mRNA和蛋白质水平研究枯草芽孢杆菌不同成分对SBD-1表达影响，以确定有效诱导成分；最后通过检测有效诱导成分的受体和信号通路各分子的变化研究其激活防御素表达的信号通路。该研究成果对揭示反刍动物胃肠道微生态黏膜免疫机制有理论意义，更为开发枯草芽孢杆菌制剂具有实践指导意义。

The emergence of antibiotic-resistant bacteria and drug residues has caused increasing harm to humans, animals and the environment. From 2020, China will comprehensively ban on adding antibiotic in feed. Defensins are a class of endogenous antibacterial peptides produced in animals. Probiotics can not only improve animal feed utilization, but also increase the production of defensins, and they are ideal substitutes for antibiotics. Yet the active ingredients and mechanisms which probiotics induce defensin expression are not completely known. It has been testified by our team that Bacillus Subtilis (BS) can improve the expression of the sheep β-defensin-1 (SBD-1) in cultured ruminal epithelium of sheep. This project firstly intends to extract the BS cell wall and culture supernatant and their components; then, the cultured sheep rumen and jejunum epithelial cells in vitro are added with different extracted components for co-culture; and the active ingredients will be identified through the qPCR and ELISA detecting the effects of different ingredients of BS on the expression of SBD-1 at the level of mRNA and protein. Finally, the signal pathways for activating defensin expression are studied by detecting changes in the receptors of the inducing ingredients and molecules of the signal pathway. The research results have theoretical significance for revealing the microecological mucosal immune mechanism of gastrointestinal tract of ruminants, and it has practical guidance significance for the development of BS preparation.