發(fā)布時間：2018-06-18 00:50

  本文選題：抗菌活性 + 生物信息學��； 參考：《青島科技大學》2014年碩士論文

【摘要】：抗生素耐藥性已成為一個全球性的公共健康問題，因此有必要開發(fā)一類新的抗菌藥物來解決這一問題。天然抗菌肽作為潛在的治療劑已經(jīng)得到了越來越多的關(guān)注�？刮⑸镫氖蔷哂袕V泛的抗微生物活性的小的陽離子多肽，廣泛存在于大多數(shù)生物體中，包括昆蟲，兩棲類，植物和哺乳動物等的先天宿主防御系統(tǒng)中，在抵御病原微生物的攻擊中發(fā)揮重要作用。雖然他們與傳統(tǒng)的抗生素相比擁有不同的作用機制，但是由于抗菌肽在應(yīng)用中出現(xiàn)的毒性，易受水解，制造成本高，尺寸和分子大小等問題，使它目前還不能進入到藥品市場中。盡管如此，抗菌肽仍然是開發(fā)無耐藥性的、安全有效的抗菌藥物的希望。因此，我們應(yīng)深入研究抗菌肽的來源和構(gòu)效關(guān)系。 雖然小的開放閱讀框（sORFs）能夠?qū)Ω鞣N生物活性肽進行編碼，但卻很少在基因組數(shù)據(jù)中被注釋出來。本研究旨在研究基礎(chǔ)脊索動物玻璃海鞘基因組序列中的sORFs編碼抗菌肽（AMPS）的潛力。首先，利用sORFfinder程序在大約4800000基因組序列中尋找潛在的sORFs，接著將sORFs翻譯成氨基酸序列，然后通過CAMP服務(wù)器預(yù)測找出最具有潛在抗菌活性的序列。最后，，選擇出10種用于抗菌肽的合成和抗微生物活性的驗證。事實上，從sORFs推導(dǎo)出180多個可能具有抗菌活性的肽。在十個實驗用肽中，我們發(fā)現(xiàn)有六個與表達序列標簽有顯著的匹配，這為基因的表達提供了有力證據(jù);有五個則被證明具有效抗菌活性。這些結(jié)果表明，海鞘基因組中的許多sORFs含有抗菌肽信息。這項研究可以作為研究sORFs在海鞘的先天防御中所起作用的一個重要的前提。 在上述合成的肽中，以P-04（含有19個氨基酸的線性陽離子肽）為模板合成了一系列N末端截短，C-端酰胺化和氨基酸取代的肽。通過對這些抗菌肽類似物進行抑菌活性研究來明確抗菌肽的構(gòu)效關(guān)系。實驗結(jié)果顯示，C-末端酰胺化增加了對大多數(shù)供試菌株的抗菌活性；N-末端的無規(guī)卷曲區(qū)域的減小也可以提高抗菌活性；丙氨酸取代絲氨酸后對大腸桿菌、變異鏈球菌和四聯(lián)球菌活性增加了2 8倍，而脯氨酸的替代導(dǎo)致了活動的明顯下降。雖然賴氨酸的取代增加了肽的凈正電荷數(shù)，但僅僅提高了對部分菌株的活性。我們對類似物的兩親性進行系統(tǒng)性的分析發(fā)現(xiàn)，抗菌活性和肽的兩親性之間并無顯著的線性關(guān)系，這表明抗菌肽要保留較高的抗菌活性就需要使這些因素之間擁有一定的平衡性。我們的研究成果能夠為新型高效抗菌肽的設(shè)計提供重要的信息。
[Abstract]:Antibiotic resistance has become a global public health problem, so it is necessary to develop a new class of antimicrobial agents to solve this problem. Natural antimicrobial peptides have attracted more and more attention as potential therapeutic agents. Antimicrobial peptides are small cationic peptides with a wide range of antimicrobial activities, which are widely found in most organisms, including insects, amphibians, plants, mammals, and other innate host defense systems. Play an important role in resisting the attack of pathogenic microorganisms. Although they have different mechanisms of action than traditional antibiotics, but because of the toxicity of antimicrobial peptides in their application, they are susceptible to hydrolysis, high manufacturing costs, size and molecular size, and so on. So that it can not enter the drug market at present. Nevertheless, antimicrobial peptides are still the hope of developing drug-resistant, safe and effective antimicrobial agents. Therefore, we should further study the origin and structure-activity relationship of antimicrobial peptides. Although small open reading frames (ORFs) can encode bioactive peptides, they are rarely annotated in genomic data. The purpose of this study was to investigate the potential of sORFs encoding antimicrobial peptides (AMPSs) in the genomic sequences of basic chordate glassy sea sheaths. First, the sORFfinder program was used to search for the potential sequence of sORFs in about 4800000 genome sequences, then the sequence of sORFs was translated into amino acid sequence, and then the sequence with the most potential antibacterial activity was predicted by the camp server. Finally, 10 kinds of antimicrobial peptides were selected for the synthesis and validation of antimicrobial activity. In fact, more than 180 peptides with antimicrobial activity were derived from sORFs. Of the ten experimental peptides, we found that six of them were significantly matched to the expressed sequence tags, which provided strong evidence for gene expression, and five were proved to be effective antimicrobial agents. These results suggest that many sORFs in the sea sheath genome contain antimicrobial peptide information. This study may serve as an important prerequisite for studying the role of sORFs in the innate defense of sea sheaths. A series of N-terminal truncated C-terminal and amino acid substituted peptides were synthesized by using P-04( a linear cationic peptide containing 19 amino acids) as template. The structure-activity relationship of antimicrobial peptides was determined by studying the antibacterial activity of these antimicrobial peptides. The results showed that amidation of C- terminal increased the decrease of random crimping region at the N- end of most strains tested, and alanine substituted serine for Escherichia coli. The activity of Streptococcus mutans and Tetraplococcus increased by 28 times, while proline substitution resulted in a significant decrease in activity. Although the substitution of lysine increased the number of net positive charges of peptides, it only increased the activity of some strains. A systematic analysis of the amphiphilic properties of analogs showed that there was no significant linear relationship between antibacterial activity and amphiphilicity of peptides. This suggests that to retain high antibacterial activity, antimicrobial peptides need to have a certain balance between these factors. Our results can provide important information for the design of new high-efficient antimicrobial peptides.
【學位授予單位】：青島科技大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R91

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