以CecMd为模板抗菌肽的新肽筛选与作用机制的研究

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.

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。

我国养殖业需要无污染的替抗产品为畜产品生产保驾护航，来满足日益提高的生活水平需要，抗菌肽CecMd对替抗有巨大的潜在优势。本研究针对CecMd抗S. aureus 活性比较弱特点，利用多种生物信息技术系统地对CecMd序列中所存在的片段进行分析，建立341条具有潜在抗菌活性肽片段库，发现其中关键片段对CecMd的活性起决定作用。在信息库中挑选5条合成进行活性分析验证了方法的可靠性，利用分子杂交设计11条肽，也验证了CecMd关键片段的起到有效抑制作用，发现CM18、CC31和CC34对S.aureus具有靶向性，并建立有效设计模式。经过毕赤酵母表达系统pGAPZA，成功表达CC29、CC30、CC34s、CecMd、CM18、Cec Md、CecMd 2ys、CecMd3cs、CecMd3js、CC313js、CC34S3js、CM18、CM184js、CC34、CC344js和CR333js，发现抗菌肽毕赤酵母聚肽化自我富集表达机制是比较有效的表达途径，同时发现抗菌肽的抑菌谱和活性强度发生改变，特别是对S.aureu，发酵的营养因素可调控表达系统蛋白生产量，最高可以达到3.84mg/ml。CecMd衍生肽体外作用机制研究发现，衍生肽有两种抑菌机制：穿膜和破膜机制。羔羊试验发现抗菌肽在肠粘膜免疫应答反应效果较好,上调小肠粘膜细胞的结构蛋白claudin-1和ZO-1的表达，下调了TLR2和TLR4等免疫细胞因子的应答反应。