抗金黄色葡萄球菌生物活性肽靶向设计及作用机制研究

Staphylococcus aureus (S. aureus) is an important pathogenic microorganism that contaminats food, though dependent on antibiotic control effect was obviously but the problems were also prominently. Therefore, it is of great scientific value to develop a new and safe bioactive peptides with antibiotic function. Previous studies have shown that it was an effective new path to create a peptides material with simulating antigen activity by anti-idiotypic antibody technology. This project is planning to choose vancomycin as an antigen mimic which is a kind of S. aureus cell wall synthesis inhibitory drug, the F(ab)2 key active fragment of its antibody was designed as a coated target, and was used for selection of anti-idiotypic bioactive peptides and genes of restrained S. aureus from the phage antibody library. The key amino acids of bioactive peptides binding to the S. aureus lipoII receptor were predicted by molecular docking, then employed the antibody modification technology for guiding to affinity maturation in vitro and obtained the higher activity target mutant peptides. Combined with molecular docking prediction, comparative analysis of the activity data of bioactive peptides by Biacore analysis and antibacterial activity test for S. aureus, then exploring the key characteristics of biological activity and resolving the mechanism of action. Finally, providing the theoretical basis and technical guarantee for the high activity and safe bioactive peptides material for restraining pathogenic microorganisms based on simulated antibiotic.

金黄色葡萄球菌（Staphylococcus aureus）是污染食品的重要致病菌，依赖抗生素防控效果显著但问题也突出，因此探索具备抗生素功能的新型安全抗菌生物活性肽具有重要的科学价值。前期研究表明，利用抗独特型抗体技术设计模拟抗原活性的多肽是一种有效的新路径。本项目拟以S.aureus细胞壁合成抑制型抗生素万古霉素为抗原模拟物，设计其抗体的F(ab)2关键活性片段作为包被靶点，从噬菌体抗体库中靶向筛选具有抗S.aureus活性的多肽及其基因；并借助分子对接及抗体改造技术对初筛多肽与模拟物受体S.aureus lipoII结合位点的关键氨基酸进行定向突变，获得高亲和力突变体；结合分子对接预测，对比Biacore分析和抗菌实验的生物活性数据，探索靶标多肽发生生物活性的关键特征规律，解析其作用机制，为高效靶向设计模拟抗生素功能的新型安全高活性的人工抗菌生物活性肽材料提供理论依据和技术保障。

金黄色葡萄球菌是污染食品的重要致病菌，依赖抗生素防控效果显著但问题也突出，因此探索具备抗生素功能的新型安全抗菌生物活性肽具有重要的科学价值。课题以抗体“免疫网络学说”中的抗独特型抗体具有模拟抗原结构和生物功能的特性为理论依据，以万古霉素为抗原模拟物，通过靶向设计体外模拟抗独特型抗体制备过程，从大容量的噬菌体展示人工多肽库中筛选具有模拟万古霉素抗金黄色葡萄球菌功能的新型安全抗菌生物活性肽材料，并结合生物信息学技术探析其活性规律，从而进一步指导抗菌生物活性肽的体外亲和力定向成熟进化，确保其应用价值。首先，课题制备出了高质量的万古霉素多克隆抗体材料，其对万古霉素的最低灵敏度达到了1.07 ng/L，为食品中万古霉素的残留检测提供了可潜在挖掘应用的高活性抗体材料。其次，课题以万古霉素多克隆抗体F(ab)2活性片段为包被靶点抗原，分别从人源和驼源噬菌体展示人工多肽库中成功获得了可模拟万古霉素抗菌功能的抗独特型生物活性肽材料30个，其中活性最强的靶标抗菌生物活性肽对金黄色葡萄球菌的最低抑菌浓度达到了125 μg/mL，初具应用研究价值。再次，通过高通量氨基酸序列比对分析，初步确定生物活性肽的CDR3区域是其与抗原和金黄色葡萄球菌lipoII受体发生结合活性的关键区域，由此指导靶标生物活性肽进行体外亲和力定向成熟进化，成功构建一个库容量为7.98×105的突变体库。最后，通过特异性生物淘筛，从突变库中成功获得高活性抗菌生物活性肽突变体材料6个，其中活性最强的突变体材料对金黄色葡萄球菌的MIC值达到了65 μg/mL，进一步推进了可应用研究的价值。课题为靶向设计抗金黄色葡萄球菌的高活性人工安全抗菌生物活性肽材料提供了理论和技术支撑。