1. 研究目的与意义
课题研究拟通过固相合成多肽，将筛选定序的多肽链化学合成纯化，并且鉴定所得活性多肽的功能。
通过抗菌实验，检验其对目标微生物金黄色葡萄球菌，大肠杆菌和白色念珠菌的抗菌能力。
该实验可以在已知并筛选出的活性多肽的基础上，通过化学合成发现新的活性多肽，通过不断修饰寻找类似物和其他活性多肽，确定其活性功能，为相关药物设计研发奠定基础。
2. 文献综述
1. IntroductionAmphibians are capable to adapt the environments filling with various microorganisms because their effective natural skin defence systems, which help them prevent from predators and microbial infections [1]. Amphibian skin is one of the most abundant sources of antimicrobial peptides (AMPs). There are about 1600 AMPs has been discovered by 2015, containing 165 species and 26 genera, and these AMPs had been arranged into at least 100 peptide families based on the sequence similarity. AMPs play important roles in native immune system and they have a wide range of antimicrobial activities which could prevent the invasions of bacteria, fungi, parasites and viruses [2]. A majority number of them show no distinct toxicity among mammalian cells [3]. AMPs have diverse primary structures and there are no specific relations among their structure, effectiveness and selectivity. Nevertheless, the key physicochemical properties of their bioactivity are size, charge, hydrophobicity and amphipathy. These AMPs share some similarities, they are normally small and amphiphilic molecules and most of them contain 8 to 45 amino acids [2]. They are mostly electropositive due to excessive residues of Arg and Lys amino acids [4], which could interact with electronegative phospholipid membranes in bacteria and/or acid bacteria membranes. Besides, their amphipathy is owing to the existence of about 50 percent hydrophobic amino acids in their sequences, which could make the membranes permeable. As a result, AMPs may have non-specific interaction with microbial membrane lipid. At the same time, the short time-frame of interaction could facilitate the rapid death of microorganisms and then lower the chances of resistance development [5]. Their antimicrobial effects are primarily accumulated on the microbial membranes, and then induce defects and disrupt the cellular osmotic gradient, and ultimately lead to cell lysis [1]. Most of them could be divided into three categories, the first group is linear α-helical peptides that do not contain cysteine residues, and the second group is those stable peptides with intramolecular disulphide bridges, the last group are certain peptides with unusual biases like histidine, glycine, proline or tryptophan [6,7]. Antimicrobial peptides, including brevinins, esculentins, temporins, dermaseptins, phylloseptins and bombinin, are capable of inhibiting the growth of some specific bacteria and fungi, and some of them may serve the function of antibiosis and/or anticancer. Phyllomedusa is a genus of leaf frogs within the hylid frog subfamily, Phyllomedusinae [8]. Phylloseptin is a recent discovered antimicrobial peptide from the skin secretion of the phyllomedusinae subfamily tree frogs. It was first reported the discovery from Phyllomedusa hypochondrialis and Phyllomedusa oreades by Leite et. al in 2005[9], Several years later, phylloseptins were isolated and identified successfully from the skin secretion of the other phyllomedusa frogs. The primary structures of phyllposepins are mainly contain 19 to 21 amino acid residues and they are positively charged with C-terminally amidated and forming α-helix domains. The bioactivity of phylloseptins is the corresponding to their physiochemical characteristics, including charge, hydrophobicity, hydrophilicity and amphiphilicity, which contributes the capability to attach the surface of the cytoplasm membranes and further interacting with membrane bilayers, resulting in the cell permeabilization. Additionally, phylloseptins show a broad spectrum antimicrobial activity on both gram-negative and positive bacteria and fungi, and a low degree of haemolytic activity, thus they are considered as more specifically targeting at prokaryotic rather than eukaryotic membranes [2,9,10]. Small molecules have rather strong pharmacological activities so they are prevalent in the drug market, while during the past few years, the ever-expanding global medicine market paid more attention to peptides due to the faster effect of them, and with the advance and breakthrough of the technology, these drugs have made significant progress in diagnosis and treatments. AMPs have been considered to be a novel therapeutic approach in the combination of conventional antibiotics in the treatment of resistant microorganisms [11]. In the meantime, the manufacturing of AMPs is more efficient than proteins-related antibiotics, such as IgM, due to a constant rate of peptide bond formation. In addition, small molecular peptides are able to spread faster than large molecular proteins and immune cells [3]. Therefore, this experiment is expected to synthetize a QUB-2054 peptide sequence by Fmoc solid-phase synthesis and verify the antimicrobial activity to S. aureus, E. coli and C. albicans by MIC and MBC measurements.
3. 设计方案和技术路线
合成多肽：运用固相多肽合成方法(SPPS)，在反应器中加入树脂作为载体并按照氨基酸已知顺序添加（序列从C端-羧基端向N端-氨基端），这些参加反应的氨基酸的侧链都已经被保护了。
脱保护和分离：利用Fmoc固相合成方法合成的多肽，在合成之后，其保护基需要被洗脱，通过加入冰乙醚三次萃取离心进行分离，最终得到成品活性多肽，上冻干机冻成粉末状，再进质谱检测。
功能筛选：将成品活性多肽应用于抗菌试验中，检验其是否具有抗菌功能。
4. 工作计划
2022030620220320 利用PubMed及Google Schoolar进行课题相关文献的检索，阅读翻译相关文献，整理综合相关知识，学习实验理论知识涉及分子生物、分子药理等；2022031320220317根据多肽序列称量准备氨基酸，利用化学生物合成多肽链；2022031820220322清洗多肽链残基，洗除保护基团，冻干成粉，质谱检测合成情况；2022032220220324利用氨基酸多肽库检索多肽序列，推测相关活性及功能；2022032820220420根据多肽序列相关活性特点，进行抗菌实验，测定最小抑菌浓度以及最小杀菌浓度；2022042220220505查补缺漏，整合实验数据，结合课题背景及相关资料完成毕业论文初稿；2022050520220515修订完善毕业论文终稿，准备模拟论文答辩。
5. 难点与创新点
从树蛙中提取的活性多肽有着很好的抗菌功能，通过固相合成的方法合成大量的天然多肽，这种方法基本原料简单易得；药效高，副作用低, 不蓄积中毒；用途广泛，品种繁多，新型药物层出不穷。
并通过MIC、MBC对天然多肽的活性功能进行检测，再进一步的修饰，使得类似物能够指导药物应用，为新型药物研发奠定基础。
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