新型抗菌肽的设计与研究—生物活性及与磷脂膜相互作用
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摘要
随着多药耐药细菌的迅速出现,发展新型的抵抗致病菌的抗菌疗法迫在眉睫。抗菌肽(Antimicrobial peptides, AMPs)是宿主先天性免疫的一部分,保护宿主免于致病细菌的入侵。抗菌肽具有广谱抗菌性,对革兰氏阴性菌、革兰氏阳性菌、病毒、真菌、原生动物以及肿瘤细胞均具有抑制作用。抗菌肽的作用机理与传统抗生素不同,因此细菌对抗菌肽不易产生抗药性。抗菌肽因其高效杀菌性、广谱抗菌性和低抗药性等特点受到广泛关注,被认为是传统抗生素的替代品或增效剂。因此,设计新型的抗菌肽,深入研究其结构特点和生物活性,筛选出具有应用潜力的抗菌肽是目前抗菌肽研究的热点之一。抗菌肽经过改造合成后,生物活性可能会得到进一步的提高。为了解决抗菌肽在应用上的诸多问题,研究设计新型抗菌肽,提高抗菌肽的杀菌活性、减少其毒性和溶血性具有重要的意义。
本文挑选多组较短序列多肽KFNFK、KFTFK、KFSFK以及RRWWRF、 FRWWHR作为活性片段,设计具有单活性序列和双活性序列的线性和环状多肽。通过研究其杀菌活性、细胞毒性、溶血性等生物活性,考察多肽结构对活性的影响。通过采用精氨酸替代赖氨酸、色氨酸替代苯丙氨酸的策略进一步设计出新的含单活性序列KWTWK、RFTFR、RWTWR的短链多肽和含有双活性序列的线性多肽,考察其生物活性,从而筛选出具有较高杀菌能力,较低细胞毒性和溶血性的多肽。此外,通过研究多肽与模拟生物膜的相互作用,探讨新型抗菌肽对生物膜的作用机理,进而为改造抗菌肽提供理论依据。
本文采用微量肉汤法测定三组人工合成抗菌肽对七种细菌的最低抑菌浓度,筛选出8条杀菌效果较好的多肽,接着采用四唑盐比色法测定8条多肽对人体正常细胞的毒性,并测得8条多肽对兔血红细胞的溶血性。生物活性实验表明,具有双活性序列的线性多肽比环状多肽、短链多肽有更显著的杀菌活性,而且对人体正常细胞毒性较小,溶血性较低,具有较理想的生物活性。活性序列加倍明显提高了多肽的杀菌活性,因此双活性序列设计有助于提高抗菌肽的杀菌性。通过考察抗菌肽作用后细菌表面膜电位的改变来反映抗菌肽对细菌细胞膜的亲和性。实验结果表明,具有双活性序列的线性多肽对细菌细胞膜的亲和性要大于短链多肽。通过考察抗菌肽与磷脂胆碱(DMPC)、磷脂酰甘油(DMPG)脂质体作用后脂质体粒径的改变研究抗菌肽对生物膜的破膜作用,结果表明所研究多肽均不能瓦解磷脂膜。采用Langmuir膜技术测量多肽1-combi-1、 c-combi-1与DMPC/DMPG单分子膜的膜压-面积曲线(π-A)曲线,对多肽-磷脂单分子膜的混合体系进行混合性分析、热力学稳定性分析及可压缩性分析。通过分析混合膜的稳定性以及分子间作用情况来反映抗菌肽插入到磷脂膜后的行为。实验结果表明,抗菌肽插入磷脂膜后与磷脂形成的混合膜的稳定性与抗菌肽的生物活性相关。
With the rapid rise in the emergence of multidrug-resistant bacteria, there is an urgent need to develop novel antimicrobial therapies to these pathogens. Antimicrobial peptides (AMPs) are an integral part of the innate immune system that protects a host from the invasion of pathogenic bacteria. Antimicrobial peptides show broad-spectrum antimicrobial activity against Gram-negative bacteria, Gram positive bacteria, viruses, fungi, protozoa and cancer cell.The mechanism of antimicrobial peptides is different from traditional antibiotics, which makes it difficult for bacteria to develop resistance. Antibacterial peptides which are characterized by high and broad-spectrum antimicrobial activity and low antibiotic resistance have attracted wide attention, and they are regarded as a substitude for traditional antibiotics or synergistic agent. Thus, one of the hot spots of the antimicrobial peptide research is to design new antimicrobial peptides, study their structures and physicochemical properties in depth and screen out the potential peptides. The modified peptides may display even higher bioactivity compared to parent peptide. In order to solve the problems of antimicrobial peptides in their application, it is of significance to design new antimicrobial peptides with increased antibacterial activity, low cytotoxicity and low hemolytic activity.
In this thesis, we chose some short sequences KFNFK, KFTFK, KFSFK and RRWWRF, FRWWHR as bioactive sequence to design short peptides with single bioactive sequence and linear and cyclic peptides with dual bioactive sequences. By studying the biological activity of these designed antimicrobial peptides, such as antibacterial activity, cytotoxicity and hemolytic acitity, the influence of peptide structure on the activity was investigated. The new short sequences KWTWK, RFTFR, RWTWR were designded based on the strategy of replacing Lysine with Arginine or substituting Tryptophan for Phenylalanine, and the new short peptides with single short sequences and linear peptides with two short sequences were further designed. Following the bioactivity investigation of these peptides, potential peptides which showed high antibacterial activity, low cytotoxicity and hemolysis were screened. In addition, the mechanism of these new antimicrobial peptides interacting with biomembrane was discussed based on the investigation of the interaction between peptides and mimic biomembrane, which provides a theoretical basis for peptide modification.
In this thesis, we used broth microdilution method to determine the minimum inhibitory concentration of the three new designed classes of peptides against seven bacteria and screened out eight peptides with high antibacterial activity. The cytotoxicity of these eight peptides against normal cell was later investigated by MTT method and their hemolytic activity was determined by hemolysis of rabbit red blood cells. These experiments show that compared with cyclic peptides and short peptides, linear peptides are more antibacterial, low cell toxic and hemolytic which indicates an ideal bioactivity. Doubling the bioactive sequence in one peptide molecule strongly increases the antibacterial activity of peptide, which suggests that dual bioactive sequences design is helpful to increase antibacterial activity of the antimicrobial peptides. Through investigating the changes of Zeta potential of bacteria after applying antimicrobial peptides on bacteria, the affinity of antimicrobial peptides on bacterial cell membrane was reflected. The experiment results show that linear peptides change the Zeta potential of bacteria to a greater extent compared with cyclic peptides and short peptides. By investigating the particle size of liposomes after antimicrobial peptides interacted with DMPC, DMPG liposomes, the disruption effect of antimicrobial peptides on biomembrane was evaluated. The results show that biological phospholipid membrane is not disrupted by all peptides. The surface pressure-area (π-A) curves of peptides1-combi-1, c-combi-1with DMPC, DMPG in mixed momolayer were measured by Langmuir technology. And then analyse the miscibility, thermodynamic stability and compressibility of the hyhrid stystem. Through analysing the stability of the mixed momolayers and the type of molecular forces to reflect the behavior after which antimicrobial peptide was inserted into the phospholipids membrane. The repulsion exists the stability of the hybrid membrane which antibacterial peptide inserted into phospholipid membrane to formed associate with the biological activity of the antibacterial peptide.
本文挑选多组较短序列多肽KFNFK、KFTFK、KFSFK以及RRWWRF、 FRWWHR作为活性片段,设计具有单活性序列和双活性序列的线性和环状多肽。通过研究其杀菌活性、细胞毒性、溶血性等生物活性,考察多肽结构对活性的影响。通过采用精氨酸替代赖氨酸、色氨酸替代苯丙氨酸的策略进一步设计出新的含单活性序列KWTWK、RFTFR、RWTWR的短链多肽和含有双活性序列的线性多肽,考察其生物活性,从而筛选出具有较高杀菌能力,较低细胞毒性和溶血性的多肽。此外,通过研究多肽与模拟生物膜的相互作用,探讨新型抗菌肽对生物膜的作用机理,进而为改造抗菌肽提供理论依据。
本文采用微量肉汤法测定三组人工合成抗菌肽对七种细菌的最低抑菌浓度,筛选出8条杀菌效果较好的多肽,接着采用四唑盐比色法测定8条多肽对人体正常细胞的毒性,并测得8条多肽对兔血红细胞的溶血性。生物活性实验表明,具有双活性序列的线性多肽比环状多肽、短链多肽有更显著的杀菌活性,而且对人体正常细胞毒性较小,溶血性较低,具有较理想的生物活性。活性序列加倍明显提高了多肽的杀菌活性,因此双活性序列设计有助于提高抗菌肽的杀菌性。通过考察抗菌肽作用后细菌表面膜电位的改变来反映抗菌肽对细菌细胞膜的亲和性。实验结果表明,具有双活性序列的线性多肽对细菌细胞膜的亲和性要大于短链多肽。通过考察抗菌肽与磷脂胆碱(DMPC)、磷脂酰甘油(DMPG)脂质体作用后脂质体粒径的改变研究抗菌肽对生物膜的破膜作用,结果表明所研究多肽均不能瓦解磷脂膜。采用Langmuir膜技术测量多肽1-combi-1、 c-combi-1与DMPC/DMPG单分子膜的膜压-面积曲线(π-A)曲线,对多肽-磷脂单分子膜的混合体系进行混合性分析、热力学稳定性分析及可压缩性分析。通过分析混合膜的稳定性以及分子间作用情况来反映抗菌肽插入到磷脂膜后的行为。实验结果表明,抗菌肽插入磷脂膜后与磷脂形成的混合膜的稳定性与抗菌肽的生物活性相关。
With the rapid rise in the emergence of multidrug-resistant bacteria, there is an urgent need to develop novel antimicrobial therapies to these pathogens. Antimicrobial peptides (AMPs) are an integral part of the innate immune system that protects a host from the invasion of pathogenic bacteria. Antimicrobial peptides show broad-spectrum antimicrobial activity against Gram-negative bacteria, Gram positive bacteria, viruses, fungi, protozoa and cancer cell.The mechanism of antimicrobial peptides is different from traditional antibiotics, which makes it difficult for bacteria to develop resistance. Antibacterial peptides which are characterized by high and broad-spectrum antimicrobial activity and low antibiotic resistance have attracted wide attention, and they are regarded as a substitude for traditional antibiotics or synergistic agent. Thus, one of the hot spots of the antimicrobial peptide research is to design new antimicrobial peptides, study their structures and physicochemical properties in depth and screen out the potential peptides. The modified peptides may display even higher bioactivity compared to parent peptide. In order to solve the problems of antimicrobial peptides in their application, it is of significance to design new antimicrobial peptides with increased antibacterial activity, low cytotoxicity and low hemolytic activity.
In this thesis, we chose some short sequences KFNFK, KFTFK, KFSFK and RRWWRF, FRWWHR as bioactive sequence to design short peptides with single bioactive sequence and linear and cyclic peptides with dual bioactive sequences. By studying the biological activity of these designed antimicrobial peptides, such as antibacterial activity, cytotoxicity and hemolytic acitity, the influence of peptide structure on the activity was investigated. The new short sequences KWTWK, RFTFR, RWTWR were designded based on the strategy of replacing Lysine with Arginine or substituting Tryptophan for Phenylalanine, and the new short peptides with single short sequences and linear peptides with two short sequences were further designed. Following the bioactivity investigation of these peptides, potential peptides which showed high antibacterial activity, low cytotoxicity and hemolysis were screened. In addition, the mechanism of these new antimicrobial peptides interacting with biomembrane was discussed based on the investigation of the interaction between peptides and mimic biomembrane, which provides a theoretical basis for peptide modification.
In this thesis, we used broth microdilution method to determine the minimum inhibitory concentration of the three new designed classes of peptides against seven bacteria and screened out eight peptides with high antibacterial activity. The cytotoxicity of these eight peptides against normal cell was later investigated by MTT method and their hemolytic activity was determined by hemolysis of rabbit red blood cells. These experiments show that compared with cyclic peptides and short peptides, linear peptides are more antibacterial, low cell toxic and hemolytic which indicates an ideal bioactivity. Doubling the bioactive sequence in one peptide molecule strongly increases the antibacterial activity of peptide, which suggests that dual bioactive sequences design is helpful to increase antibacterial activity of the antimicrobial peptides. Through investigating the changes of Zeta potential of bacteria after applying antimicrobial peptides on bacteria, the affinity of antimicrobial peptides on bacterial cell membrane was reflected. The experiment results show that linear peptides change the Zeta potential of bacteria to a greater extent compared with cyclic peptides and short peptides. By investigating the particle size of liposomes after antimicrobial peptides interacted with DMPC, DMPG liposomes, the disruption effect of antimicrobial peptides on biomembrane was evaluated. The results show that biological phospholipid membrane is not disrupted by all peptides. The surface pressure-area (π-A) curves of peptides1-combi-1, c-combi-1with DMPC, DMPG in mixed momolayer were measured by Langmuir technology. And then analyse the miscibility, thermodynamic stability and compressibility of the hyhrid stystem. Through analysing the stability of the mixed momolayers and the type of molecular forces to reflect the behavior after which antimicrobial peptide was inserted into the phospholipids membrane. The repulsion exists the stability of the hybrid membrane which antibacterial peptide inserted into phospholipid membrane to formed associate with the biological activity of the antibacterial peptide.
引文
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