多股β-发卡抗菌肽的分子设计、生物活性及作用机理的研究

【作者】 丑淑丽； 单安山；

【机构】 东北农业大学动物科学技术学院；

【摘要】 抗菌肽（Antimicrobial peptides,AMPs）,作为一类新型的抗生素替代物,主要包括了四种结构,而这四种结构对于抗菌肽本身生物学活性的发挥又是至关重要的。相对于其他结构的抗菌肽,β-折叠结构抗菌肽因其具有较高的抗菌活性及细胞选择性,具有更大的开发前景。此外,有研究表明增加β-折叠个数所得的"S-状"结构模型具有更高的结构稳定性。因此,我们期望通过设计一系列含有多个转角的抗菌肽获得具有更高稳定性的抗菌肽,即多股β-发卡。并在此结构的基础上,利用色氨酸拉链提高结构稳定性及抗菌活性,设计得到了一系列全新结构的抗菌肽（（WRXxRW）n-NH₂,其中n=1、2、3or4;Xx代表转角氨基酸）,并从中筛选出抗菌活性高、序列长度短的抗菌肽。首先,利用圆二色谱及红外光谱检测此系列肽在模拟膜环境中或水溶液环境中的结构构象。抑菌试验采用最小抑菌浓度（MIC）指标采判定。肽的安全性通过溶血活性来验证。随后利用扫描电镜,透射电镜来探讨肽的抑菌机理。结果表明,所有抗菌肽都能达到60%以上的β-折叠（转角）结构倾向。此外,二倍体抗菌肽（n=2）表现出较强的抗菌活性,且对正常细胞无溶血活性,对于所测试的所有菌株,W2（WR.PGRW）2具有更高的抗菌活性。在此基础上,电镜试验结果表明W2可以通过破坏细菌细胞膜导致其内容物流出而达到杀菌的目的。因此,基于多股β-发卡结构,W2凭借其经济、高效以及安全性高的优势具有更好的应用潜力。更多还原

【Abstract】 Antimicrobial peptides(AMPs) are a potential new class of antibacterial agents.They mainly present four structures,and the secondary structures are very important to the biological activity of the antibacterial peptide itself.Compared with other structures,the p-sheet structure peptides have been proposed to exhibit potent antimicrobial properties without cytotoxicity,thus,they were developed as functional AMPs.Along with that multiple p-hairpin structures,which are likely as S-shaped motif,are proved to be more stability and form tightly packed hydrophobic core.In addition,the tryptophan zipper(Trp-Zip) was applied to increase the activity and the stability further.Thus the aim of this study was to develop peptide-based synthetic strategies for generating short,but effective AMPs as inexpensive antimicrobial agents.Multiple-stranded P-hairpin peptides with the same Trp-Zip P-hairpin unit,(WRXxRW)n where n= 1,2,3 or 4 and Xx represents the turn sequence,were synthesized,and their application potential was evaluated.The results showed that the tendency to be P-sheet/turn of all the peptide are more than 60%,and the 12-residues peptides exhibited high cell selectivity towards bacterial cells over human red blood cells(hRBCs),and the peptide W2 exhibited stronger antimicrobial activities with the MIC values of 2-8 uM against various tested bacteria.Electron microscopy analyses indicated that W2 kills microbial cells by permeabilizing the cell membrane and damaging membrane integrity.Collectively,based on the multiple P-hairpin structure peptides,the ability to develop libraries of short and effective peptides will be a powerful approach to the discovery of novel antimicrobial agents.更多还原