开菲尔源干酪乳杆菌L04抗菌肽的结构表征及关键基因调控机制研究

The antimicrobial peptides produced by lactic acid bacteria is important in the application for food and medicine industry. It is necessary to clarify the structure, antimicrobial mechanism and regulatory mechanism of key gene cluster of antimicrobial peptides, which will provide the theoretical basis for the development and utilization of antimicrobial peptides. The crude polypeptide (<30 kDa), produced by the strain Lactobacillus casei L04 isolated from Tibetan kefir, exerted inhibiting effect on Escherichia coli, Staphylococcus aureus and Bacillus cereus. But the structure, antimicrobial mechanism and biosynthetic gene cluster of the crude peptide are not clear. The purpose of the project is to study the separation and purification of crude peptides produced by Lb. casei L04 to get single peptide component; study the antimicrobial effect and mechanism of antimicrobial peptides; study the primary structural characteristic of antimicrobial peptides composition including determining the amino acids composition and sequence, the linkages of peptides bond, branched-chain, and so on; study the advanced structral characteritic of antimicrobial peptides including the molecular spiral and folding; study the key genes which involve in the antimicrobial peptides synthesis by the bioinformatics analysis of Lb. casei L04 genome and gene knockout techniques.

乳酸菌抗菌肽在食品及医药领域有着广泛的应用前景，阐述抗菌肽的结构、抑菌机理及关键基因调控机制，将为抗菌肽的开发利用提供理论依据。本课题组从西藏开菲尔中分离获得产抗菌肽的干酪乳杆菌L04，前期研究发现该菌株产生的粗多肽（<30 kDa）能够抑制大肠杆菌、金黄色葡萄球菌和蜡样芽胞杆菌等实验菌的生长，但其结构、抑菌机理及合成基因簇尚不明确。本项目拟对该菌株粗多肽进行分离纯化和抑菌性研究，获得抗菌肽单一组分。研究抗菌肽的抑菌机理；研究抗菌肽组分的氨基酸组成和排列方式、肽键连接形式、支链情况等，确定抗菌肽分子螺旋、折叠程度等，明确其一级结构和高级结构；对干酪乳杆菌L04进行生物信息学分析和基因敲除实验，定位具有抑菌作用的抗菌肽单一组分生物合成基因簇，分析基因序列并进行功能注释。

抗菌肽（Antimicrobial peptides，AMPs）是一类具有巨大发展潜力的新型食品用防腐剂。乳酸菌抗菌肽是在乳酸菌代谢过程中，合成并分泌到环境中的一类对微生物具有抑制作用的杀菌蛋白或多肽。分离具有强抑菌效果的菌株，并从中提取纯化抗菌肽，研究抗菌肽的化学组成、结构与抑菌机理之间的关系，是天然防腐剂发展的重要途径之一。运用选择性培养基结合革兰氏染色以及过氧化氢酶接触实验，筛选出乳酸菌，并通过牛津杯抑菌法初步筛选出具有抑菌活性的乳酸菌；再进行有机酸和H2O2排除实验，以及蛋白敏感实验来确定抑菌物质为蛋白类。通过生理生化和16S rDNA鉴定菌种并构建系统发育树。测定其抑菌谱尤其是对食源性腐败菌和致病菌的抑菌效果。将分离得到的强抑菌效果菌株进行发酵，随后用PREP-HPLC对抗菌肽进行纯化，并进行Tricine-PAGE凝胶电泳后进行液质连用质谱分析，对其进行定性鉴定。利用纯化的抗菌肽作用于荧光假单胞菌以研究其抑菌机理。抗菌肽处理荧光假单胞菌后胞内核酸、钾离子等物质的外泄，推测该抗菌肽的作用机制可能是通过作用于指示菌细胞膜，最终引起菌体死亡。进行全基因组测序后分析，BAGEL数据库预测该抗菌肽结构基因的区域。