基于海洋生物基因组信息的抗菌肽的设计和功能研究

发布时间：2018-06-18 00:50

本文选题：抗菌活性 + 生物信息学　；参考：《青岛科技大学》2014年硕士论文

【摘要】：抗生素耐药性已成为一个全球性的公共健康问题，因此有必要开发一类新的抗菌药物来解决这一问题。天然抗菌肽作为潜在的治疗剂已经得到了越来越多的关注。抗微生物肽是具有广泛的抗微生物活性的小的阳离子多肽，广泛存在于大多数生物体中，包括昆虫，两栖类，植物和哺乳动物等的先天宿主防御系统中，在抵御病原微生物的攻击中发挥重要作用。虽然他们与传统的抗生素相比拥有不同的作用机制，但是由于抗菌肽在应用中出现的毒性，易受水解，制造成本高，尺寸和分子大小等问题，使它目前还不能进入到药品市场中。尽管如此，抗菌肽仍然是开发无耐药性的、安全有效的抗菌药物的希望。因此，我们应深入研究抗菌肽的来源和构效关系。虽然小的开放阅读框（sORFs）能够对各种生物活性肽进行编码，但却很少在基因组数据中被注释出来。本研究旨在研究基础脊索动物玻璃海鞘基因组序列中的sORFs编码抗菌肽（AMPS）的潜力。首先，利用sORFfinder程序在大约4800000基因组序列中寻找潜在的sORFs，接着将sORFs翻译成氨基酸序列，然后通过CAMP服务器预测找出最具有潜在抗菌活性的序列。最后，，选择出10种用于抗菌肽的合成和抗微生物活性的验证。事实上，从sORFs推导出180多个可能具有抗菌活性的肽。在十个实验用肽中，我们发现有六个与表达序列标签有显著的匹配，这为基因的表达提供了有力证据;有五个则被证明具有效抗菌活性。这些结果表明，海鞘基因组中的许多sORFs含有抗菌肽信息。这项研究可以作为研究sORFs在海鞘的先天防御中所起作用的一个重要的前提。在上述合成的肽中，以P-04（含有19个氨基酸的线性阳离子肽）为模板合成了一系列N末端截短，C-端酰胺化和氨基酸取代的肽。通过对这些抗菌肽类似物进行抑菌活性研究来明确抗菌肽的构效关系。实验结果显示，C-末端酰胺化增加了对大多数供试菌株的抗菌活性；N-末端的无规卷曲区域的减小也可以提高抗菌活性；丙氨酸取代丝氨酸后对大肠杆菌、变异链球菌和四联球菌活性增加了2 8倍，而脯氨酸的替代导致了活动的明显下降。虽然赖氨酸的取代增加了肽的净正电荷数，但仅仅提高了对部分菌株的活性。我们对类似物的两亲性进行系统性的分析发现，抗菌活性和肽的两亲性之间并无显著的线性关系，这表明抗菌肽要保留较高的抗菌活性就需要使这些因素之间拥有一定的平衡性。我们的研究成果能够为新型高效抗菌肽的设计提供重要的信息。
[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.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R91

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