本文選題：抗菌肽 + 構(gòu)效關(guān)系　； 參考：《重慶理工大學(xué)》2017年碩士論文

【摘要】：全球每年的細菌感染病患高達15億人,其中死亡人數(shù)460萬,特別是多藥耐藥菌(MDRB)感染導(dǎo)致的高死亡率,嚴重威脅公眾健康;歐洲與美國每年因細菌感染防治的支出高達70億歐元和65億美元,而發(fā)展中國家則需為此承擔更為高昂的代價�？咕�(AMP)是生物體為抵御外源性病原菌入侵而產(chǎn)生的一類多肽物質(zhì),也是自然免疫的重要組成部分,具有陽離子性和兩親性的基本結(jié)構(gòu)特征。AMP與傳統(tǒng)抗生素相比,具有抗菌譜廣、殺菌快速、耐藥性低等傳統(tǒng)抗生素無法比擬的系列優(yōu)點,有望開發(fā)成為一類高效、低毒的新型抗菌藥物。本文以現(xiàn)有小分子抗菌活性多肽為基礎(chǔ),使用計算機輔助藥物設(shè)計方法建立定量預(yù)測模型,確定AMP的優(yōu)勢位點與優(yōu)勢氨基酸;以牛乳鐵蛋白片斷LfcinB64-9(RRWQWR)為模板,結(jié)合抗菌肽的結(jié)構(gòu)特征與定量預(yù)測模型研究結(jié)果,設(shè)計并篩選獲得3條抗菌活性較高、溶血活性低、細胞毒性小、穩(wěn)定性好的小分子抗菌肽。本研究主要開展了如下工作:1、從多肽數(shù)據(jù)庫(APD2)和文獻中收集抗菌多肽序列,基于氨基酸保守序列分析與構(gòu)效關(guān)系研究,確定AMP序列模式、優(yōu)勢位點及優(yōu)勢氨基酸,以LfcinB64-9為模板,結(jié)合AMP序列特征,虛擬組合設(shè)計小分子AMP;2、利用定量預(yù)測模型篩選獲得14條候選小分子抗菌肽,使用固相合成方法合成抗菌肽及其模板,所有抗菌肽純度達到95%以上,并由質(zhì)譜鑒定結(jié)構(gòu);3、對小分子抗菌肽進行系列生物活性實驗驗證,主要包括抑菌試驗、紅細胞溶血毒性試驗、細胞毒性實驗、血漿中穩(wěn)定性試驗、脂質(zhì)膜溶解實驗,確定AMP的抗菌效果以及毒性效應(yīng),為下一步成藥研究奠定基礎(chǔ)。本研究所取得的主要成果如下:1、建立了小分子抗菌肽組合設(shè)計、虛擬篩選、定量預(yù)測、活性驗證的完整技術(shù)方案,為小分子AMP的設(shè)計與改造奠定了基礎(chǔ)。2、本研究篩選獲得了3條(RWRWRW、RRWWRF、RRWWRW)抗菌活性高、毒副作用小、穩(wěn)定性好的小分子抗菌肽,其中RRWWRW的對鮑曼不動桿菌具有良好抑菌效果,其最小抑菌濃度為16μg/ml,而且在512μg/ml時的細胞毒性與紅細胞溶解活性低于5%。本研究建立AMP組合設(shè)計與虛擬篩選的理論預(yù)測模型,為小分子抗菌肽的合理設(shè)計與高效篩選提供完整技術(shù)方案,篩選獲得1個對耐藥飽滿不動桿菌抗菌活性強、穩(wěn)定性好、選擇性高的小分子抗菌肽,為新型低耐藥性多肽抗菌藥物的設(shè)計與開發(fā)奠定基礎(chǔ)。同時,利用計算機輔助藥物設(shè)計方法指導(dǎo)抗菌肽的設(shè)計與篩選,不僅能夠獲得結(jié)構(gòu)多樣的高活性抗菌肽分子,而且可以提高篩選速度,減少研究成本。
[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.
【學(xué)位授予單位】：重慶理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R91;R96

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相關(guān)期刊論文 前1條

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本文編號：2049498