畜禽致腹泻大肠杆菌抗体及可变区导向的专一性抗菌肽设计与作用机理

With the increasing Escherichia coli infection leading to diarrhea and the extensive use of feed antibiotics in animal husbandry, the resistance of pathogens toward antibiotics become serious. Antimicrobial peptides (AMPs) are the ideal substitute for antibiotics. However, the balance of microecology in the host would also be disrupted due to its wide antimicrobial spectrum rsulting from physical destructure of membranes. As the result, few AMPs were used in the animal husbandry. Therefore, the development of targeted antimicrobial peptides will be conducive to the health of livestock. Based on the foundation of our lab and the tendence of the animal husbandry, the aim of this work will be focused on the following three aspects: 1) the specific antibody and its variable region to the LPS of E. coli O149 causing the diarrhea in animal husbandry would be screened and combined with typical AMPs -- Lactoferricin B and Arenicin to design targeted antimicrobial peptide sequences. Additionally, the biochemical parameters of theses AMPs would be optimized and evaluated by using the bioinformatics tools. These studies will provide a theoretical basis and experiential patterns for the design of new targeted AMPs; 2) the specificity of targeted AMPs would be studied by testing the bactericidal properties to the target strains, control strains and probiotics. In addition, bactericidal efficiency of targeted AMPs and the capacity of binding and neutralizing endotoxin would be also evaluated. Furthermore, the bactericidal mechanism of the targeted AMPs would be determined by using the ELISA and electron microscopy technique; 3) The chemical solid-phase synthesis and the Pichia pastoris expression system would be combined in a rational way to develop the preparation system which specially for the small molecular weight AMPs. In conclusion, all of above works will lay the foundation for the development of specific targeted antimicrobial peptides and it will also be helpful for the prevention and treatment of E. coli infection in animal husbandry.

长期采用饲料抗生素应对大肠杆菌感染引起的畜禽腹泻问题，导致病原菌耐药性日益严重，还因广谱杀菌性严重破坏了肠道微生态平衡。抗菌肽是有潜力的抗生素替代品之一，本申请拟创制具有高度特异性的靶向抗菌肽克服上述挑战。研究内容：1）针对最常见的畜禽致腹泻性大肠杆菌O149，研制其LPS特异结合抗体及可变区，再与具临床应用潜力抗菌肽Lactoferricin B、Arenicin连接，利用生物信息学工具建立靶向抗菌肽关键参数的评价、筛选等设计原则和经验模型，设计针对大肠杆菌O149的靶向抗菌肽；2）实证确认靶向抗菌肽专一性，体内外实验评价其杀菌效率及其与内毒素结合解毒的能力。利用ELISA和电镜等技术探讨其抗菌机制；3）将化学固相合成和毕赤酵母转基因表达有机结合，建立适用于小分子靶向抗菌肽的高效制备系统和关键技术。意义：该新型靶向抗菌肽在大肠杆菌引起的畜禽腹泻防治中的应用，将促进饲料抗生素的减量使用。

本项目针对目前畜禽养殖中常规抗生素治疗大肠杆菌感染存在耐药性与引发内毒素释放等问题，按照合同任务书要求，开展以下研究：1）通过生物信息学方法对大肠杆菌外膜蛋白进行分析，锚定OmpA、BamA、TolC、OmpC及OmpF进行保守性筛选、结构性质及免疫原性生物信息学分析；2）OmpA、BamA、TolC、OmpC及OmpF的基因克隆、重组表达与纯化；3）OmpA、BamA、TolC、OmpC及OmpF通用亚单位疫苗的体内、体外评价试验；4）OmpA单链可变区抗体获取、构建及表达；5）OmpC和OmpF蛋白单链抗体核糖体展示文库的构建；6）与LPS特异结合抗体及可变区连接的海蚯蚓肽及乳铁蛋白肽抗菌机理研究。.完成以下研究目标：1）建立了一套基于理化性质、保守性、抗原性、同源性及系统进化分析的亚单位疫苗的设计体系；2）建立了一套大肠杆菌外膜蛋白抗原表位疫苗的设计以及大肠杆菌单链可变区抗体获取、构建及表达方法，为革兰氏阴性细菌抗体药物研发奠定了基础；3）创制了OmpA、BamA、TolC、OmpC及OmpF等五种通用型亚单位疫苗；4）建立了一套大肠杆菌外膜蛋白单链抗体核糖体展示文库的构建方法；5）建立了一套基于流式细胞仪、圆二色谱、扫描电镜和透射电镜等的抗菌肽的机制研究体系。6）发表论文26篇（SCI论文25篇，中文核心期刊论文1篇）；7）申请专利12项（授权专利5项）；8）培养博士后1名，培养博士生1名，培养硕士生4名；9）获北京市科技进步奖一等奖1项，第十八届中国国际高新技术成果交易会优秀产品奖1项。.本项目为病发快、来势重、难控制的烈性病原菌传染病防治提供了新方法和新思路，为今后疫苗发展的新方向。