新型双肽乳酸菌细菌素PLNC8抗菌靶点及分子作用机制研究

PLNC8 is a Class IIb lactic acid bacteria (LAB) bacteriocin with great thermal stability and antibacterial activity and can effectively inhibit both Gram-positive and Gram-negative food-borne pathogens. The latest study in our lab found that, different from Nisin, the target of PLNC8 is not Lipid II but cell membrane protein. Identification the target of PLNC8 and uncovering its antibacterial mechanism would provide basic knowledge for its application in food industry. In this project, a screening system combining GST Pull-down and iTRAQ-LC-MS/MS was established to efficiently search for candidate target proteins. Heterologous expression, binding assay and fluorescence leakage assay were used to determine the target protein and then elucidate the antibacterial mechanism of PLNC8. The findings in this proposal will not only provide insight into the mechanism of the novel LAB bacteriocin PLNC8, but also provide a view to the use of PLNC8 for food (preservatives) applications.

PLNC8为ClassⅡb类细菌素，热稳定、抗菌活性强，能高效抑制多种革兰氏阳性和阴性食源性病原菌，作为新型的乳酸菌源食品抗菌剂有很好的应用前景。确立该细菌素抗菌分子机制将为其开发应用提供生物学基础。本课题组最新研究发现PLNC8有别于Nisin，对敏感菌藤黄微球菌的作用靶点不是LipidⅡ而是细胞膜蛋白。本研究拟建立ClassⅡb细菌素作用靶点鉴定新体系筛选PLNC8作用靶点：建立GST Pull-down和iTRAQ-LC-MS/MS相结合的筛选体系寻找候选靶蛋白，运用异源表达、结合试验、荧光泄露试验来确定作用靶点，初步阐明PLNC8抗菌分子机制。项目的完成有助于更全面系统地认识ClassⅡb类乳酸菌细菌素抗菌作用机制，可为该细菌素在食品工业中的应用提供坚实的理论基础。

ClassⅡb类细菌素PLNC8，能够高效抑制多种革兰氏阳性和阴性食源性病原菌，具有抑菌谱较广、热稳定性高等特点，作为新型的乳酸菌源食品抗菌剂，具有较好的开发及应用前景。为探究该细菌素的抗菌分子机制，本课题组采用藤黄微球菌作为指示菌，研究PLNC8的抗菌作用模式。提取藤黄微球菌细胞膜蛋白，并在大肠杆菌中异源表达融合蛋白。以GST Pull-down和iTRAQ-LC-MS/MS融合的技术建立筛选体系，通过高浓度细菌素筛选抗性突变体，寻找候选靶蛋白，利用结合试验、荧光泄露试验对潜在作用靶点进行表征。根据PLNC8的靶点蛋白筛选方法，建立ClassⅡb细菌素作用靶点鉴定新体系。本项目的完成有助于进一步系统地认识ClassⅡb类乳酸菌细菌素抗菌作用机制，可为该细菌素在食品工业中的应用打下坚实的理论基础。