丁酸梭菌与猪肠粘膜细胞互作过程中磷壁酸的作用解析及其受体鉴定

项目名称： 丁酸梭菌与猪肠粘膜细胞互作过程中磷壁酸的作用解析及其受体鉴定

项目编号： No.31272461

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 王进波

作者单位： 浙江大学宁波理工学院

项目金额： 60万元

中文摘要： 本项目以丁酸梭菌为模型，探究磷壁酸在菌体粘附、定植及免疫调节中的作用，并鉴定其受体蛋白。采用基因同源重组技术敲除磷壁酸合成关键酶tagO基因，获得磷壁酸合成缺陷突变体△tagO，进而以荧光标记技术研究磷壁酸在丁酸梭菌粘附、定植过程中的作用；以生物信息学方法模拟筛选猪肠粘膜细胞膜上的磷壁酸受体，运用基因定点突变和绿色荧光蛋白标记技术，鉴定猪肠粘膜细胞膜上与磷壁酸互作的受体蛋白，从而为阐明丁酸梭菌粘附、定植的分子机制提供新线索。综合运用ELISA、Western-blotting、Real time PCR和RNAi技术，通过对猪肠粘膜细胞NF-kB和ERK信号通路的研究，探究磷壁酸在丁酸梭菌免疫调节过程中的作用，并阐明其分子机制，为揭示肠道益生菌和致病菌的免疫刺激机制提供新思路，进而为益生菌在动物营养领域的开发、利用提供技术基础和理论依据。

中文关键词： 丁酸梭菌；脂磷壁酸；仔猪；肠道；功能

英文摘要： In this study, the roles of teichoic acid will be investigated during Clostridium butyricum adhesion,colonization and immune regulation. The receptors of teichoic acid also will be identified. The tagO gene of Clostridium butyricum will be knockd out by homologous recombination technique, the tagO mutant deficient in teichoic acid will be constructed and then fluorescence labeling technique will be used to study the function of teichoic acid during Clostridium butyricum adhesion and colonization. The receptors of teichoic acid will be screened with bioinformatics methods and then the receptors will be identified by Site-directed Mutagenesis and Enhanced Green Fluorescent Portein (EGFP) Labeling techniques, which will provide new clues for understanding the mechanism of Clostridium butyricum adhesion and colonization. ELISA,western-blotting, real time PCR and RNAi will be applied to study the effects of teichoic acid from Clostridium butyricum on the NF-κB and ERK signaling pathways, which will supply a new idea for revealing the immune stimulation mechanisms of probiotics and pathogenic bacteria. This study will provide technical and theoretical basis for the research and development of probiotics in animal nutrition.

英文关键词： Clostridium Butyricum；Lipoteichoic acid；piglet；intestine；function