乳酸杆菌S-层蛋白拮抗PEDV感染宿主细胞机制的研究

Porcine epidemic diarrhea virus (PEDV) enter and invade host cell by adhesion、cell internalization、cytoskeleton disruption、interference of cell apoptosis pathways in their hosts. Inhibiting PEDV adhesion is effective to protect host cell against invasion. PEDV are major virus causing diarrhea in young pigs and led to an important economic losses. According to the fact that the antagonistic properties of food Lactic acid bacteria (Lactobacillus). The project is going to study the inhibitory mechanism of Lactobacillus S-layer proteins against to the PEDV. Firstly the S-layer proteins are purified, the interaction of S-layer proteins and PEDV are detected. Thereafter, the influence of S-layer proteins on cultured cells of intestine are measured in vitro and vivo respectively, including the virus adhesion、the cytoskeleton、the factors in cell apoptosis pathways in cultured cells. At last the local immunity in intestine is detected. The results will reveal the mechanism of antagonistic properties of Lactobacilli S-layer proteins on virus, provide a model for mechanism of other pathogens, as well as lead to new preventive and therapeutic strategies for the diarrhea in young pigs.

PEDV（Porcine epidemic diarrhea virus，PEDV）主要是通过黏附、细胞内化、改变细胞微丝骨架结构和影响细胞凋亡的达到入侵宿主上皮细胞的目的。拮抗和阻止病毒的黏附作用是保护细胞免受侵害的有效方法。PEDV是引起仔猪腹泻的主要病原微生物。根据乳酸杆菌通过S-层蛋白抑制PEDV黏附达到拮抗作用的现象，本项目拟研究乳酸杆菌S-层蛋白拮抗PEDV的作用机制。首先提取和纯化乳酸杆菌的S-层蛋白，检测乳酸杆菌S-层蛋白与PEDV的相互作用；然后通过体外和体内试验分别检测S-层蛋白抑制PEDV对小肠细胞的影响，包括病毒的黏附入侵、细胞骨架的变化、细胞凋亡通路中几种因子的变化等；最后检测小肠局部免疫功能的变化。本项目的结果不仅揭示猪小肠宿主乳酸杆菌S-层蛋白拮抗PEDV的机制，还对研究其他病原微生物致病机制提供理想模型，可为预防和治疗仔猪腹泻提供理论基础。

首先提取和纯化嗜酸乳酸杆菌的S-层蛋白，研究S-层蛋白对PEDV（Porcine epidemic diarrhea virus，PEDV）的拮抗作用；然后应用Vero细胞作为体外模型，检测S-层蛋白拮抗PEDV黏附及入侵Vero细胞的影响，包括PEDV的黏附入侵、细胞微丝的变化、细胞凋亡中几种因子的变化和体内免疫反应的影响等。结果显示：1）在竞争、排斥、置换三种黏附试验中，S-层蛋白仅在排斥试验中发挥拮抗作用，S-层蛋白不影响PEDV的黏附，可能抑制PEDV的入侵和复制。2）S-层蛋白能够拮抗PEDV对Vero细胞微丝的破坏，可能通过上调Rac1蛋白发挥作用。3）S-层蛋白可抑制Vero细胞Caspase-8和Caspase-3的活化，拮抗PEDV引起的细胞凋亡损伤。4）S-层蛋白可显著增加小肠上皮内淋巴细胞的数量。. 结果表明：S-层蛋白可能通过拮抗PEDV对细胞微丝的破坏；通过抑制Caspase-8和Caspase-3的活性，拮抗PEDV引起的细胞凋亡损伤；上调上皮内淋巴细胞的募集，共同介导拮抗PEDV的感染。