禽类防御素AvBD103b干扰病原菌细胞膜和核酸的关键结构研究

项目名称： 禽类防御素AvBD103b干扰病原菌细胞膜和核酸的关键结构研究

项目编号： No.31302004

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 农业科学

项目作者： 滕达

作者单位： 中国农业科学院饲料研究所

项目金额： 24万元

中文摘要： 在课题组禽类β-防御素AvBD103研究的前期工作基础上，开展如下研究：采用截短二级结构区或氨基酸替换破坏结构区的方法来研究AvBD103b的α-螺旋倾向N端、β-折叠、Loop结构及疏水斑对病原菌细胞膜和核酸的干扰作用。具体是用透射电镜观察细胞膜变化、与脂质体互作、外膜及质膜渗透性测定、胞内钾离子泄露实验和细菌细胞膜完整性分析进行AvBD103b对细菌细胞膜作用的研究；通过DNA凝胶阻滞实验、原子力显微镜扫描与DNA结合、圆二色谱检测DNA结构变化、竞争性结合荧光光谱以及与DNA特异性结合分析进行AvBD103b对细菌DNA或RNA的作用的研究，通过上述结果明确AvBD103b对细胞膜和核酸干扰的关键结构，揭示AvBD103b的抗菌机理，进一步完善禽类β-防御素的抗菌机理，为推动禽类防御素AvBD103b作为饲料添加剂在替代抗生素方面的应用奠定理论基础。

中文关键词： 禽类防御素；AvBD103b；肠炎沙门氏菌；抗菌机理；关键结构

英文摘要： On the basis of our research group previous work about avian beta-defensin, more theoretical and experimental research will be conducted as follows: The method of the truncation of the secondary structur and amino acid substitution in the secondary stucture including N-terminal with α-helix propensity, β-sheet, loop structure and hydrophobic patch were used to find these structures how to interfere with the cell membrane and nucleic acids of the pathogen. The method of membrane changes observed by transmission electron microscopy, interaction with liposomes, determination of the outer membrane and the plasma membrane permeability, leakage of intracellular potassium ion experiments and bacterial cell membrane integrity analysis were used to analysis the effection of AvBD103b on bacterial cell membrane. The method of DNA gel retardation assay, scanning DNA binding by atomic force microscope, detection of DNA structural changes by circular dichroism, competitive binding fluorescence spectroscopy and specific binding to DNA analysis were used to analysis the effection of AvBD103b to bacterial DNA or RNA. The key structure interfering with the cell membrane and nucleic acids and anti-bacterial mechanism of AvBD103b will be clarified and the antimicrobial mechanism of avian β-defensin will be further improvend, which

英文关键词： Avian defensin；AvBD103b；Salmonella enteritidis；antibacterial mechanism；key structure