【作者】 冉瑜；

【导师】 屈凌波；

【作者基本信息】 郑州大学 ， 分析化学， 2013， 硕士

【摘要】 抗菌肽的主要作用位点是细菌细胞膜的骨架——磷脂双分子层,由于磷脂酰胆碱(DMPC)和磷脂酰甘油(DMPG)分别为构成动植物细胞膜和细菌细胞膜的重要磷脂,因此本论文分别采用DMPC和DMPG来模拟动植物细胞膜和细菌细胞膜,研究抗菌肽对不同细胞的破膜效果。本研究从头设计了三组新型抗菌肽,并采用电化学法,荧光淬灭法以及理论计算模拟的方法对三组抗菌肽与磷脂之间的相互作用进行了研究,研究结果如下：采用KFNFK、KFSFK、KFTFK为活性片段设计含有单活性序列的短链多肽、含有双活性序列的线型肽和环状肽,并对该组多肽和磷脂的相互作用进行了研究。计算模拟的结果表明,双活性序列抗菌肽能够提供较单活性序列肽多的正电荷氨基酸与DMPG负电头基形成静电吸引,因此具有较高的结合能,而环状肽因为其较为固定的构型和序列较短使得其结合能低于线型肽。以固体支撑双层膜模拟生物膜,采用电化学方法研究了所设计多肽与磷脂之间的相互作用,结果显示,抗菌肽无论与DMPC还是DMPG进行作用,具有双活性序列的抗菌肽较单活性序列抗菌肽更易对磷脂发生破坏作用；抗菌肽对DMPG的破坏作用的程度较DMPC更易受到抗菌肽浓度的影响。采用从抗菌肽库中搜索所得到的短链多肽RRWWRF和FRWWHR为活性序列设计含单活性序列的短链肽和含双活性序列的线型肽和环状肽,并对多肽与磷脂的相互作用进行研究。理论计算的结果显示,双活性序列抗菌肽的结合能大于单活性序列抗菌肽,双活性序列抗菌肽更易于DMPG结合；电化学实验结果显示,第二组抗菌肽只能对DMPC产生破坏作用,而对DMPG基本上不具有破坏作用。因此推测第二组抗菌肽并不是通过对细菌细胞膜产生破坏作用而杀菌,而是与细菌细胞膜上的磷脂产生相互作用并进入到细胞膜内部,与细胞内物质产生相互作用而使细菌死亡。采用RFTFR、RWTWR、KWTWK为活性片段设计含有单活性序列的短链多肽和含有双活性序列的线型肽,并对该组多肽和磷脂的相互作用进行了研究。计算模拟的结果显示双活性序列的抗菌肽具有较单活性序列抗菌肽高的结合能,更易与DMPG结合。电化学实验结果显示,双活性序列抗菌肽能够与DMPC和DMPG发生作用使膜破裂,而单活性序列抗菌肽则基本不与DMPC和DMPG产生作用；抗菌肽对DMPG的破坏作用较DMPC大。荧光淬灭实验结果显示,短链肽基本不能插入到DMPG和DMPC产生相互作用,而线型肽均能插入到DMPG中却不能插入到DMPC中。更多还原

【Abstract】 The most important property of antimicrobial peptide is their special sterilization mechanism. The antimicrobial peptide mainly targets the backbone of bacterial cell membrane-phospholipid bilayer. Because DMPC and DMPG are the main phospholipids to constitute the membrane of flora and fauna and bacterium respectively, the DMPC and DMPG are used to simulate the flora and fauna and bacterium cell membrane to investigate the interaction between antimicrobial peptides with different phospholipids. Three groups of antimicrobial peptides are designed in this research. The interaction between these three groups peptide and phospholipids are investigated by electrochemistry, fluorescence quenching and compute simulation method. The results are as follows.The sequences KFNFK, KFSFK, KFTFK were chosen in the first group as single bioactive sequence, and according to these sequences, linear and cyclic peptides with dual bioactive sequences were designed and the interaction between peptides and phospholipids were studied. The result of compute simulation showed that peptides with dual bioactive sequences could offer more positive charged amino acids than short peptides with single bioactive sequence to facilitate the electrostatic attraction with negative charged head group of DMPG, which leads to higher binding energy with DMPG for linear and cyclic peptides. Because of the rigid configuration and short sequence of cyclic peptides, so they have a lower binding energy with DMPG compared to linear peptides. The interaction between peptides and phospholipid was further investigated by electrochemistry method by using solid supported bilayer as membrane mimic. The results showed that peptides with dual bioactive sequences have a higher destructive effect against phospholipid bilayer than peptide with single bioactive sequence regardless of DMPC or DMPG. The influence of peptides concentration on the interaction with DMPG was more sensitive than DMPC.RRWWRF and FRWWHR which are from antimicrobial peptides database were chosen as single bioactive sequences for designing linear and cyclic antimicrobial peptides with dual bioactive sequences, and their interaction with phospholipids was further investigated. Compute simulation results showed that peptides with dual bioactive sequences had a higher binding energy with DMPG than peptides with single bioactive sequence, which indicates that peptides with dual bioactive sequences are easier to bind to DMPG. The electrochemistry experiment results showed that this second group peptide couldn’t destruct DMPG membrane though they had a destructive effect to DMPC. So we deduced that the second group peptides could interact with bacterial cell membrane, enter the interior of cell, interact with the substance in cell and finally kill the bacterium, instead of disrupting cell membrane.Peptides with single bioactive sequence and dual bioactive sequences using RFTFR, RWTWR, KWTWK as bioactive segments has been designed and their interaction with phospholipids was further studied. The compute simulation results showed that linear peptides with dual bioactive sequences had higher binding energy than peptides with single bioactive sequence, indicating a stronger binding of linear peptides with DMPG. The electrochemistry experiment results showed that peptides with dual bioactive sequences could disrupt DMPC or DMPG membrane while peptides with single bioactive sequence hardly disrupt phospholipid membrane. Peptides showed stronger disrupting ability on DMPG membrane compared to DMPC membrane. The results of fluorescence quenching experiment showed that short peptides hardly inserted into DMPC or DMPG lipsomes. However linear peptides could inserted into DMPG lipsomes but nearly have no interaction with DMPC.更多还原