小分子抗菌肽的设计、合成、与生物活性研究

发布时间：2018-06-21 16:34

本文选题：抗菌肽 + 构效关系　；参考：《重庆理工大学》2017年硕士论文

【摘要】：全球每年的细菌感染病患高达15亿人,其中死亡人数460万,特别是多药耐药菌(MDRB)感染导致的高死亡率,严重威胁公众健康;欧洲与美国每年因细菌感染防治的支出高达70亿欧元和65亿美元,而发展中国家则需为此承担更为高昂的代价。抗菌肽(AMP)是生物体为抵御外源性病原菌入侵而产生的一类多肽物质,也是自然免疫的重要组成部分,具有阳离子性和两亲性的基本结构特征。AMP与传统抗生素相比,具有抗菌谱广、杀菌快速、耐药性低等传统抗生素无法比拟的系列优点,有望开发成为一类高效、低毒的新型抗菌药物。本文以现有小分子抗菌活性多肽为基础,使用计算机辅助药物设计方法建立定量预测模型,确定AMP的优势位点与优势氨基酸;以牛乳铁蛋白片断LfcinB64-9(RRWQWR)为模板,结合抗菌肽的结构特征与定量预测模型研究结果,设计并筛选获得3条抗菌活性较高、溶血活性低、细胞毒性小、稳定性好的小分子抗菌肽。本研究主要开展了如下工作:1、从多肽数据库(APD2)和文献中收集抗菌多肽序列,基于氨基酸保守序列分析与构效关系研究,确定AMP序列模式、优势位点及优势氨基酸,以LfcinB64-9为模板,结合AMP序列特征,虚拟组合设计小分子AMP;2、利用定量预测模型筛选获得14条候选小分子抗菌肽,使用固相合成方法合成抗菌肽及其模板,所有抗菌肽纯度达到95%以上,并由质谱鉴定结构;3、对小分子抗菌肽进行系列生物活性实验验证,主要包括抑菌试验、红细胞溶血毒性试验、细胞毒性实验、血浆中稳定性试验、脂质膜溶解实验,确定AMP的抗菌效果以及毒性效应,为下一步成药研究奠定基础。本研究所取得的主要成果如下:1、建立了小分子抗菌肽组合设计、虚拟筛选、定量预测、活性验证的完整技术方案,为小分子AMP的设计与改造奠定了基础。2、本研究筛选获得了3条(RWRWRW、RRWWRF、RRWWRW)抗菌活性高、毒副作用小、稳定性好的小分子抗菌肽,其中RRWWRW的对鲍曼不动杆菌具有良好抑菌效果,其最小抑菌浓度为16μg/ml,而且在512μg/ml时的细胞毒性与红细胞溶解活性低于5%。本研究建立AMP组合设计与虚拟筛选的理论预测模型,为小分子抗菌肽的合理设计与高效筛选提供完整技术方案,筛选获得1个对耐药饱满不动杆菌抗菌活性强、稳定性好、选择性高的小分子抗菌肽,为新型低耐药性多肽抗菌药物的设计与开发奠定基础。同时,利用计算机辅助药物设计方法指导抗菌肽的设计与筛选,不仅能够获得结构多样的高活性抗菌肽分子,而且可以提高筛选速度,减少研究成本。
[Abstract]:There are 1.5 billion patients with bacterial infection in the world every year. Among them, 4.6 million people die, especially the high mortality rate caused by MDRBB infection, which is a serious threat to public health. Europe and the United States spend 7 billion euros and $6.5 billion a year on the prevention and treatment of bacterial infections, and developing countries have to pay more. Antimicrobial peptide (AMP) is a kind of polypeptide produced by organisms to resist the invasion of exogenous pathogens, and it is also an important part of natural immunity. AMP has the basic structural characteristics of cationic and amphiphilic. AMP is compared with traditional antibiotics. With the advantages of wide antibacterial spectrum, rapid sterilization and low drug resistance, it is expected to be a new class of antibiotics with high efficiency and low toxicity. In this paper, a quantitative prediction model of AMP was established by computer aided drug design based on the existing peptides with antibacterial activity. The dominant sites and amino acids of AMP were determined, and the fragment of bovine lactoferrin LfcinB64-9 RRWQWR was used as template. Based on the structural characteristics of antimicrobial peptides and the results of quantitative prediction model, three antimicrobial peptides with high antibacterial activity, low hemolytic activity, low cytotoxicity and good stability were designed and screened. The main work of this study was to collect antimicrobial peptide sequences from peptide database APD2) and literature. Based on the analysis of amino acid conserved sequence and structure-activity relationship, AMP sequence patterns, dominant sites and dominant amino acids were determined, and LfcinB64-9 was used as template. Combined with AMP sequence characteristics, a virtual combination was designed for the design of small molecule AMPX 2. Fourteen candidate antimicrobial peptides were selected by quantitative prediction model. The antibacterial peptides and their templates were synthesized by solid phase synthesis. The purity of all antimicrobial peptides was over 95%. A series of bioactivity tests were carried out to verify the bioactivity of small molecular antimicrobial peptides, including bacteriostasis test, erythrocyte hemolytic toxicity test, cytotoxicity test, plasma stability test, lipid membrane dissolution test. The antimicrobial and toxic effects of AMP were determined to lay a foundation for the further study of proprietary medicine. The main achievements of this study are as follows: 1. A complete technical scheme for the combination design, virtual screening, quantitative prediction and activity verification of small molecular antimicrobial peptides was established. In this study, three small molecular antimicrobial peptides with high antimicrobial activity, low toxicity and good stability were obtained. RRWWRW had a good antibacterial effect on Acinetobacter baumannii, and RRWWRW had good antimicrobial activity against Acinetobacter baumannii. The minimal inhibitory concentration was 16 渭 g / ml, and the cytotoxicity and erythrocyte lytic activity were lower than 5 at 512 渭 g/ml. In this study, the theoretical prediction model of AMP combination design and virtual screening was established, which provided a complete technical scheme for the rational design and efficient screening of small molecular antimicrobial peptides, and obtained one with strong antibacterial activity and good stability against acinetobacter sativum resistance. The small molecular antimicrobial peptides with high selectivity lay the foundation for the design and development of novel low-resistance polypeptide antimicrobial agents. At the same time, using computer-aided drug design method to guide the design and screening of antimicrobial peptides, not only can obtain a variety of highly active antimicrobial peptide molecules, but also can improve the screening speed and reduce the research cost.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91;R96

【参考文献】