饲用抗菌肽MP1102对产气荚膜梭菌的防御机理研究

On the basis of our research group previous work about antibacterial peptides, more theoretical and experimental research on the defense mechanism of feeding antimicrobial peptide MP1102 against Clostridium perfringens for its harm reduction in animal husbandry will be conducted as follows: 1) The method of minimum inhibitory concentration, minimum bactericidal concentration, time-killing kinetic curve and combination index will be used to analyse in vitro antimicrobial characteristic. 2) The antimicrobial in vivo characteristic will be studied by therapeutic dose, pathological tissue investigation, cell factor analysis, pharmacokinetics and postantibiotic postantibiotic effect assay in infected mouse model. 3) Feeding effectiveness trials will be performed on piglets and broiler. 4) The method of transmission electron microscope, laser scanning confocal microscope, atomic absorption spectroscopy, flow cytometry, gel retardation assay, scanning DNA binding by atomic force microscope, detection of DNA structural changes by circular dichroism will be used to analysis the effection of MP1102 to the cell membrane and intracellular DNA or RNA of Clostridium perfringens. The above results will provide the support of pharmacodynamics and pharmacokinetic background data of MP1102 in the prevention of Clostridium perfringens and the technical applications reference of MP1102 in animal feed, and the antibacterial mechanism will be clarified, which will lay theoretical and application foundation for promotting MP1102 as a new type of antimicrobial agent on the alternative to antibiotics.

在课题组饲用抗菌肽研究前期工作基础上，针对畜牧业中危害极大的产气荚膜梭菌，开展新型饲用抗菌肽MP1102对产气荚膜梭菌防御机理的如下研究：1）通过最小抑菌/抑菌浓度、杀菌动力学、联合用药、溶血性及细胞毒性等测定进行MP1102体外抗菌的表征研究；2）通过小鼠产气荚膜梭菌感染模型进行治疗剂量、组织病变观察、细胞因子分析、药代动力学、抗生素后效应等研究明确其体内抗菌表征；3）进行仔猪和肉鸡的有效性饲喂试验；4）借助透射电镜、激光共聚焦显微镜、原子吸收光谱、流式细胞术、凝胶阻滞、原子力显微镜和圆二色谱等技术分析MP1102对细胞膜和胞内核酸的干扰作用。上述研究为饲用抗菌肽MP1102在产气荚膜梭菌预防中提供药效、药代学背景数据支撑；为MP1102在畜禽饲料中添加提供配套应用技术参考；明确MP1102作用靶点，揭示其抗菌机理；为推动MP1102作为新型抗菌制剂在替代抗生素方面奠定理论和应用基础。

抗生素在畜牧业中过量使用导致病原菌耐药性增强，禁止其在饲料中添加已成为国际共识，因此，寻求安全、高效的新型抗菌制剂已经迫在眉睫。防御素以其独特的抑菌机制、抗菌谱广、不易产生耐药性以及来源安全等优点，有可能被开发成传统意义上的抗生素替代品。菌丝霉素衍生肽以其特异性高效杀灭革兰氏阳性菌而可能成为新型高效的抗菌制剂的候选制剂。但在其对产气荚膜梭菌的体外抗菌表征、抗菌机理以及防治模式动物和靶动物感染有效性等方面的研究目前还处于空白，这些极大限制了菌丝霉素衍生肽成为新型抗生素替代品在饲料上的应用。本项目（1）通过最小抑菌/抑菌浓度、杀菌动力学、联合用药等体外抗菌表征MP1102和NZ2114对产气荚膜梭菌具有杀菌活性强、速度快等特点，优于林可霉素、乳酸链球菌素、金霉素和维吉尼亚霉素；（2）通过对小鼠大腿感染模型和肉鸡坏死性肠炎模型证明MP1102和NZ2114可以有效治疗产气荚膜梭菌感染的小鼠和肉仔鸡，效果优于林可霉素；（3）用透射电镜观察细胞膜变化及流式细胞仪分析碘化丙啶染色细菌胞膜完整性等发现MP1102和NZ2114对产气荚膜梭菌壁膜具有干扰作用，通过破坏膜结构，造成细胞内容物泄漏，导致细胞死亡；（4）凝胶阻滞、圆二色谱以及流式细胞仪等实验结果表明胞内DNA也是MP1102和NZ2114的作用靶点，其能够与DNA结合并影响DNA的复制和细胞的分裂。上述研究为饲用抗菌肽MP1102和NZ2114在产气荚膜梭菌预防中提供药效学背景数据支撑；为其在畜禽饲料中添加提供配套应用技术参考；明确MP1102和NZ2114作用靶点，揭示其抗菌机理；为推动MP1102和NZ2114作为新型抗菌制剂在替代抗生素方面奠定理论和应用基础。.合同之外，将菌丝霉素衍生肽NZ2114作为另一个投入品做上述研究。