1. 研究目的与意义
内容：1. 通过固相化学合成法合成多肽。
2. 通过高效液相色谱法进行纯化。
3. 通过质谱法进行结构验证。
2. 文献综述
Introduction:The global resumption of infectious diseases has sustained over three decades for the unfolding of the antibiotic-resistant strains, as the potency of antibiotics weakened increasingly. So it is urgent for people to get the substitution of the antibiotics [1]. Also, In the past 35 years It has been reported that the peptides/proteins seem to have a better performance in diagnostics and therapeutics of than the small molecules drug for its technical breakthrough and development, and the worldwide pharmaceutical market has put an increasing money to the peptide [2]. So the research to search substitution of antibiotics from peptide is likely to make sense. And The connection between amphibian and the environment inspire human beings a lot. In order to survive preferably in the hostile nature, amphibians have schemed successfully. Though it seems that most anurans would have a weaker ability of valid physiological control because of their evaporative water loss (EWL) from the integument. So most of them must live close to or direct in water and have special behavior, such as work nocturnally and keep wet by fossorial activity. However, in this case, microorganisms may attach to the skin of frogs and toads for their physiological secretions for benefit. To find an environment-survival balance, the secretions may constitute by some kind of natural compounds that could protect the animal from microorganisms [3]. Actually, the idea has been verified by human beings, and it is proved that the antimicrobial peptides are created constantly through secretion holocrine mechanisms and keep in the granular glands of skin [4]. So they are adequate in amphibians for protect their nude skin and wound from illness and infection. Also there are many types of antimicrobial peptides in the secretions of amphibians [5]. The discovery and exploring of Amphibian antimicrobial peptides has been an intense subject for many years. And plenty of them have been separate from amphibians [3]. Many of them have characteristic including α-helical and amphipathic of structure, cationic property, antifungal and antibacterial activities in a large spectrum and so on [6]. For the mechanisms of majority of these antimicrobial peptides, they are thought to attach to the surface of microbial membrane by drawing support from the cationic property, and practice the permeability in virtue of the amphipathic helical structure. Ultimately, destroy the cytoplasmic membrane of target microorganisms [7,8]. So, rather than coupled with specific receptors or targets, these antimicrobial peptides tend to less resistance. Also, both the interaction with membranes and the action goes inside the cell could practice the antimicrobial potent. [7]Antimicrobial peptides from amphibian constitute by a large family. And peptide from Hylids could be divided precisely into 7 families by multiple comparison analysis, including the dermaseptins, the phylloseptins, the plasticins, the dermatoxins, the phylloxins, the hyposins, the orphan peptides [9].Phylloseptins (PS) are a family of antimicrobial cationic peptides discovered in Phyllomedusa genus (anura), from the secretions of the tree frogs living in the tropical forests of South and Central America. They are amphipathic peptides that made up from 19-21 highly conserved amino acid residues and show an C-terminal amidation [1,5,7]. Most peptides of this family have histidine in sequence at number 7 and 18. Considering both structure and functions, most of them possess high similarity in the N-terminal region, and a high level of alteration come to the last six residues [4]. The amphiphilic theme of numerous antimicrobial peptides could be confirmed by its three-dimensional structures. The hydrophilic part is constituted by residues that is polar and with positive charge and the hydrophobic part is made up by non-polar amino acids [1]. And resemble other cationic antimicrobial peptides, they show a wide range of antibacterial activities including counterwork gram-negative and gram-positive bacteria (Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, Candida albicans, Streptococcus agalactiae) and yeast (Candida albicans), specific sequence of amino acid stay connected with the detailed spectrum of the antibacterial and antimycotic activities. [4]Apart from all this goodness, some difficulties and disadvantages of these peptide drugs limit their application in clinic, which need to take measures urgently. In the way of oral, which delivery with gastrointestinal proteases, degradation peptide drugs easily. Also, they would be sensitive enough to degradation by enzymatic, though they have already come into the systemic circulation. So it would lead to a low bioavailability, and therapeutic index could be easily influenced as well. Consequently, practicing the stability and meanwhile the bioavailability of these antimicrobial peptide has been one of the theme of the future research. On the other hand, antimicrobial peptides have several other shortcomings such as high costs and short half-lives. However, continuity of efforts in exploration, improvement and innovation would get the batter of these shortcomings [2]. And the antimicrobial peptides whose potent has been evaluated critically may become a type of new antibiotics that could rescue the crisis of worldwide health in a way. [10]In this work, we expand experiments centre on a novel antimicrobial peptide which belong to Phylloseptin family. And we get the peptide by the method of chemically-synthesis, determine the sequence through mass spectrometry and affirm the antibacterial effect with staphylococcus aureus, escherichia coli and Candida albicans.Reference:1. Jos Roberto S.A. Leite, Luciano P. Silva, Maria Izabel S. Rodrigues, Maura V. Prates, Guilherme D. Brand, Bruno M. Lacava, Ricardo B. Azevedo, Anamlia L. Bocca, Sergio Albuquerque, Carlos Bloch Jr., Phylloseptins: a novel class of anti-bacterial and anti-protozoan peptides from the Phyllomedusa genus, Peptides, Volume 26, Issue 4, April 2005, Pages 565-573, ISSN 0196-9781.2. Yitian Gao, Di Wu , Xinping Xi , Yue Wu , Chengbang Ma , Mei Zhou , Lei Wang , Mu Yang , Tianbao Chen and Chris Shaw, Identification and Characterisation of the Antimicrobial Peptide, Phylloseptin-PT, from the Skin Secretion of Phyllomedusa tarsius, and Comparison of Activity with Designed, Cationicity-Enhanced Analogues and Diastereomers, Molecules 2016, 21(12), 1667.3. Katia Conceio, Katsuhiro Konno, Michael Richardson, Marta M. Antoniazzi, Carlos Jared, Sirlei Daffre, Antonio Carlos M. Camargo, Daniel C. Pimenta, Isolation and biochemical characterization of peptides presenting antimicrobial activity from the skin of Phyllomedusa hypochondrialis, Peptides, Volume 27, Issue 12, December 2006, Pages 3092-3099, ISSN 0196-9781.4. Selma Kckelhaus, Jos Roberto, S. A. Leite, Mateus, P. Neves, Toxicity Evaluation to Mice of Phylloseptin-1, an Antimicrobial Peptide from the Skin Secretion of Phyllomedusa hypochondrialis (Amphibia), September 2007, Volume 13, Issue 3, pp 423429.5. Selma A.S. Kckelhaus, Jos Roberto S.A. Leite, Maria Imaculada Muniz-Junqueira, Raimunda Nonata Sampaio, Carlos Bloch Jr., C. Eduardo Tosta, Antiplasmodial and antileishmanial activities of phylloseptin-1, an antimicrobial peptide from the skin secretion of Phyllomedusa azurea (Amphibia), Experimental Parasitology, Volume 123, Issue 1, September 2009, Pages 11-16, ISSN 0014-48946. Yasser H.A. Abdel-Wahab, Gavin J. Power, Peter R. Flatt, Douglas C. Woodhams, Louise A. Rollins-Smith, J. Michael Conlon, A peptide of the phylloseptin family from the skin of the frog Hylomantis lemur (Phyllomedusinae) with potent in vitro and in vivo insulin-releasing activity, Peptides, Volume 29, Issue 12, December 2008, Pages 2136-2143, ISSN 0196-9781.7. Jarbas M. Resende, Rodrigo M. Verly, Christopher Aisenbrey, Amary Cesar, Philippe Bertani, Dorila Pil-Veloso, Burkhard Bechinger, Membrane Interactions of Phylloseptin-1, -2, and -3 Peptides by Oriented Solid-State NMR Spectroscopy, Biophysical Journal, Volume 107, Issue 4, 19 August 2014, Pages 901-911, ISSN 0006-3495.8. Raja Z, Andr S, Piesse C, Sereno D, Nicolas P, et al. (2013) Structure, Antimicrobial Activities and Mode of Interaction with Membranes of Bovel Phylloseptins from the Painted-Belly Leaf Frog, Phyllomedusa sauvagii. PLOS ONE 8(8): e70782. Doi.9. Mohamed Amiche, Ali Ladram, Pierre Nicolas, A consistent nomenclature of antimicrobial peptides isolated from frogs of the subfamily Phyllomedusinae, Peptides, Volume 29, Issue 11, November 2008, Pages 2074-2082, ISSN 0196-9781.10. Xinping Xi, Renjie Li, Yingchun Jiang, Yan Lin, Yuxin Wu, Mei Zhou, Jie Xu, Lei Wang, Tianbao Chen, Chris Shaw, Medusins: A new class of antimicrobial peptides from the skin secretions of phyllomedusine frogs, Biochimie, Volume 95, Issue 6, June 2013, Pages 1288-1296, ISSN 0300-9084.中文：三十多年来，随着耐药菌株对药物的的进一步耐受，抗生素的效力逐渐减弱，全球传染病一直在持续，人们正迫切地需要抗生素的替代品[1]。
另外，据报道，在过去的35年中，由于技术的发展和突破，肽/蛋白质在诊断和治疗方面对比小分子药物有较好的优势，全球医药市场对肽和蛋白质的投资比例呈上升的趋势[2]。
因此，从肽中寻找抗生素的替代品具有一定意义。
3. 设计方案和技术路线
使用天然的L型氨基酸以标准的Fmoc固相化学合成的方法合成多肽，脱保护基，洗脱，得到较纯的多肽。
进一步以HPLC纯化，通过质谱法验证多肽序列，通过金黄色葡萄球菌，大肠杆菌和白色念珠菌的抗菌实验进行功能筛选。
4. 工作计划
2022.3.13: 按照序列内容称取氨基酸3.20: 称取树脂, 并进行多肽的化学合成3.28: 对合成结束的肽进行去除保护基，洗脱3.29: 冻干3.5-3.28: 阅读文献3.29: 完成开题报告4.3-4.5: 下冻干并进行抗菌实验
5. 难点与创新点
采用固相合成的方法合成大量的天然多肽。
使连接在固相载体(树脂)上的氨基酸与溶解在有机溶剂中的氨基酸之间反应。
因反应在一简单反应器中便可进行；固相载体共价相联的肽链处于适宜的物理状态，可通过快速的抽滤、洗涤未完成中间的纯化，避免了液相肽合成中冗长的重结晶或分柱步骤，可避免中间体分离纯化时大量的损失；使用过量反应物，迫使个别反应完全，以便最终产物得到高产率。
以上是毕业论文开题报告，课题毕业论文、任务书、外文翻译、程序设计、图纸设计等资料可联系客服协助查找。
