以CecMd为模板抗菌肽的新肽筛选与作用机制的研究

项目名称： 以CecMd为模板抗菌肽的新肽筛选与作用机制的研究

项目编号： No.31472120

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 畜牧学与草地科学

项目作者： 姜宁

作者单位： 黑龙江八一农垦大学

项目金额： 86万元

中文摘要： CecMd是从家蝇幼虫脂质体表达的抗菌肽，含40个氨基酸残基的阳离子抗菌肽，对E. coli、S. Salomon和S. dysgalactiae都有不同程度的抑杀作用，但其抗S. aureus活性比较弱。本研究拟利用生物信息学和分子生物学技术重新整合CecMd信息，优化序列，引入对S. aureus抗性强的氨基酸序列，筛选出活性高、谱宽、溶血活性更低，对S. aureus抗性更强的CecMd衍生肽。在此基础上，通过荧光显微镜、电镜扫描、分子生物学等技术研究抗菌肽CecMd衍生肽与靶向细菌膜的作用及CecMd衍生肽与靶向细菌DNA和RNA的作用；以羔羊为模式动物，通过PCR-DGGE技术和免疫学技术研究CecMd衍生肽对羔羊肠道菌群的调控作用和对羔羊肠道免疫组化因子、肠道上皮免疫应答效应器TLRs的分析，揭示CecMd与衍生肽的抑菌机制。为抗菌肽饲料添加剂的研制、开发和利用提供有效的依据。

中文关键词： 抗菌肽；定向进化；基因表达；微生物多样性；羔羊

英文摘要： The antimicrobial peptide (AMP) Cec Md is cecropins analogue from the lipidsome of Musca domestica larva, which is cationic peptide contains 40 amino acids. We investigated the activity of Cec Md resistant to E. coli、S. Salomon and S. dysgalactiaebefore. Compared to amphillicin, the antibacterial activity of Cec Md is stronger, however, it is weaker when the Cec Md interacted with S. aureus. Our purpose is screening two or more new peptides with more active and lower hemolytic based on the Cec Md template chain. By bioinformatic, molecular biological technology, we will integrate Cec Md segment through DNA shuffling, insert one short sequence with stronger resistance to S. aureus through screening from libirary of ADP or database published. We intend to gain further insight into the mechanisms of action between AMPs and cellular membranes using target E. coli and S. aureus by scanning electron scope and transmission electron microscope, the interaction between AMPs and DNA, RNA of target bacteria will also be determined by molecular technology. By PCR-DGGE, immunohistochemical technology and immunological molecular technology and lamb as model animal, we will carry out the analysis of the bateria colony in gastrointestine, distribution of mast cell, Goblet cell, intraepithelial lymphocyte and secretory immunological antibody in epithelium in order that reveal mechanism of the novel peptides intact microorganism in gastrointestine, and the mRNA expression of TLRs will be determined as a immunological respose effector to different bacteria under AMPs regulating in vivo. Elucidating the mechanism of the membranolytic activity of AMPs may facilitate the development of more effective antimicrobial agents.

英文关键词： Antimicrobial peptide;site-mutangenesis;gene expresion;microbial flora;lamb