本文選題：抗菌肽 + LfcinB64-9虛擬組合設計�。� 參考：《重慶理工大學》2012年碩士論文

【摘要】：針對當前抗菌藥物耐藥性強的問題，特別是抗生素的濫用日趨嚴重，人類亟待開發(fā)出一類新型、高效、低毒、無殘留的抗菌藥物。而抗菌肽具有抗菌譜廣、穩(wěn)定性好、副作用小、無耐藥性等優(yōu)點，有望克服當前抗菌藥物的耐藥性強、副作用大等系列難題。目前，多種分子設計與改造方法用于抗菌肽研究，期望在提高抗菌活性同時進一步降低毒副作用，進而為抗菌肽的臨床應用奠定基礎；盡管現(xiàn)有抗菌肽的分子改造取得了可喜進展，但在抗菌活性、化學合成、細胞毒性等方面還存在諸多問題。 基于上述研究現(xiàn)狀，本文提出全新“肽鏈短、電荷高、兩親性強、優(yōu)勢氨基酸多”的“小分子“”陽離子”抗菌肽的設計理念和虛擬組合設計方法。以陽離子抗菌肽LfcinB64-9(RRWQWR)為模板，基于抗菌肽模式序列要求、優(yōu)勢位點的氨基酸結(jié)構及其化學修飾，開展抗菌肽的虛擬組合設計，并通過定量構效關系實現(xiàn)其高通量篩選，具體如下：①對現(xiàn)有抗菌肽數(shù)據(jù)庫(APD2)中的抗菌肽進行一級序列比對分析，確定陽離子抗菌肽的序列模式；②對現(xiàn)有陽離子抗菌肽進行定量構效關系研究，確定核心序列的優(yōu)勢氨基酸及其化學修飾方法；③以LfcinB64-9為模板，基于序列模式、優(yōu)勢位點的氨基酸及其化學修飾，虛擬產(chǎn)生結(jié)構多樣的抗菌肽庫；④基于構效關系分析及擬肽二級結(jié)構篩選進行合成；⑤采用Fmoc固相方法合成擬肽、RP-HPLC純化并質(zhì)譜鑒定；⑥擬肽與LfcinB64-9通過抗菌活性、細胞毒性、穩(wěn)定性考查篩選所設計的最好抗菌肽，為下一步的成藥奠定基礎。 本文主要的研究成果是：(1)建立小分子陽離子抗菌肽的組合虛擬篩選路線：(2)成功的篩選到三條高效、低毒、副作用小、無耐藥性的小分子陽離子抗菌活性多肽。實驗結(jié)果表明“肽鏈短、電荷高、兩親性強、優(yōu)勢氨基酸多”的小分子陽離子抗菌肽的設計理念和虛擬組合設計方法，節(jié)約了新型抗菌肽設計與篩選的時間和成本，并提高了抗菌肽分子設計的合理性和可行性。本文所設計、篩選的三條抗菌肽有望成為結(jié)構特異性好、體內(nèi)抗酶解能力強、作用機理明確、毒副作用小、化學合成容易、具有成藥潛力的小分子陽離子抗菌肽。 總而言之，基于組合肽庫與分子模擬方法開展抗菌肽的虛擬組合設計與高通量篩選研究，不僅可以基于構效關系研究成果合理設計結(jié)構多樣性的多肽分子，，并有效提高篩選效果、降低研究成本；有望進一步提高其抗菌活性并解決規(guī)模生產(chǎn)問題，對抗菌肽走向?qū)嶋H臨床應用具有重要理論研究價值。
[Abstract]:In view of the problem of strong antimicrobial resistance, especially the serious abuse of antibiotics, it is urgent to develop a new class of antimicrobial drugs with high efficiency, low toxicity and no residue. However, antimicrobial peptides have the advantages of wide antibacterial spectrum, good stability, small side effects and no drug resistance, which are expected to overcome the current antimicrobial drug resistance, side effects and other problems. At present, many molecular design and modification methods are used in the study of antimicrobial peptides, in order to improve the antibacterial activity and further reduce the toxic side effects, thus laying a foundation for the clinical application of antimicrobial peptides. Although the molecular modification of antimicrobial peptides has made great progress, there are still many problems in antibacterial activity, chemical synthesis, cytotoxicity and so on. Based on the above research status, this paper presents a new "small molecule" cationic "antimicrobial peptide design method with" short peptide chain, high charge, strong amphiphilicity and many dominant amino acids ". The cationic antimicrobial peptide LfcinB64-9 (RRWQWR) was used as the template. Based on the requirements of the antibacterial peptide pattern sequence, the amino acid structure of the dominant site and its chemical modification, the virtual combination design of the antimicrobial peptide was carried out, and its high throughput screening was realized by quantitative structure-activity relationship. The following is the first order sequence alignment analysis of antimicrobial peptides in the existing antimicrobial peptide database (APD2), and the quantitative structure-activity relationship of existing cationic antimicrobial peptides is determined by determining the sequence pattern of cationic antimicrobial peptides. To determine the dominant amino acids of core sequences and their chemical modification methods: using LfcinB64-9 as template, based on sequence patterns, amino acids at dominant sites and their chemical modification, the antibacterial peptide libraries with various structures were produced. 4 based on the structure-activity relationship analysis and the screening of the secondary structure of the mimic peptide, the synthesis method was used to purify the peptide by FMOC solid phase method by RP-HPLC and to identify the antibacterial activity and cytotoxicity of LfcinB64-9 and the peptide by mass spectrometry. Stability test and screening of the best antimicrobial peptides designed to lay the foundation for the next step of proprietary medicine. The main results of this paper are as follows: (1) to establish a virtual screening route for small cationic antimicrobial peptides; (2) to successfully screen three small cationic antimicrobial peptides with high efficiency, low toxicity, small side effects and no drug resistance. The experimental results show that the design concept and virtual combination design method of small cationic antimicrobial peptides with short peptide chains, high charge, strong amphiphilic properties and more dominant amino acids can save the time and cost of the design and screening of new antimicrobial peptides. The rationality and feasibility of antimicrobial peptide design were improved. The three antimicrobial peptides designed in this paper are expected to be small cationic antimicrobial peptides with good structural specificity, strong anti-enzymatic ability in vivo, clear mechanism, small toxic side effects and easy chemical synthesis. In a word, virtual combinatorial design and high-throughput screening of antimicrobial peptides based on combinatorial peptide library and molecular simulation methods can not only reasonably design polypeptide molecules with diverse structures based on the results of structure-activity relationship research. It is expected to further improve its antibacterial activity and solve the problem of large-scale production, which has important theoretical research value for the practical clinical application of antimicrobial peptides.
【學位授予單位】：重慶理工大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

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