1. 研究目的与意义
抗生素的发现和应用在治疗病原性传染病和保护人体健康方面发挥了重要作用。
然而，由于抗生素的广泛使用甚至滥用，细菌耐药性，过敏反应，环境污染等问题日益严重。
因此，人们开始高度重视食品和环境安全，寻找新的抗菌剂。
2. 文献综述
IntroductionThe discovery and application of antibiotics have played a significant role both in the treatment of pathogenic infectious diseases and protection of human health. Nevertheless, with the wide application even abuse of antibiotics, the problem of bacterial resistance, anaphylaxis, environment pollution and other issues have become increasingly serious. As a result, people begin to pay high attention to food and environment safety, and are looking for novel antimicrobial agents. Antimicrobial peptides discovered in recent years are one of the new antimicrobial drugs with high development potential.Antimicrobial peptides encoded by genes exist widely in insects, plants, animals and human bodies, which are major components of congenital immune defense system. They possess broad spectrum of antimicrobial activity, especially have notable bacteriostatic and bactericidal effects for multidrug-resistant bacteria [1]. Furthermore, antimicrobial peptides have good thermal stability and water solubility and nearly have no toxic effects on the normal cells of higher animals. Therefore, antimicrobial peptides have become hot spots for research and development throughout the world.Antimicrobial peptides could be divided into 5 categories on the basis of their structures: linear peptides with helical structures, linear peptides rich in some amino acids, peptides containing a disulfide bond, peptides containing two or more disulfide bonds and peptides derived from larger peptides with known functions. Although antimicrobial peptides have various structures, practically all of them share some common characteristics. Natural antimicrobial peptides are generally small molecule cationic polypeptides consisting of 12-60 amino acids. They usually contain 2-7 positive charges and isoelectric points are over 7, showing strong cationic features. It is possibly because that they have large quantities of lysine, arginine and histidine and other basic amino acids. Amphiphilic structures are the common structures of antimicrobial peptides. The hydrophobic regions combine with the lipid and the hydrophilic regions with positive charges combine with the water or the negative residues. These features make antimicrobial peptides attach well with cell membrane composed of amphiphilic molecules. Moreover, they act well especially for cell membrane with negative charges, and this is the structure foundation of antimicrobial peptides interacting with cell membrane [2]. One of the potential antimicrobial peptides sources are amphibian skin secretions. A variety of amphibians live in moist and moggy habitats. These environments make them face high risk of infection. They have to secrete antimicrobial substances so that they could get rid of infection and diseases. Many amphibians could synthesize and secrete antimicrobial peptides with broad spectrum antibacterial, antifungal and other activities according to their own needs [3]. Phyllomedusinae is a subfamily of hylid tree frogs comprises 57 species in seven genera, scilicet Agalychnis, Cruziohyla, Hylomantis, Pachymedusa, Phasmahyla, Phrynomedusa and Phyllomedusa [4]. From the family of these antimicrobial peptides, Phylloseptins are widely found from the skin secretions of Phyllomedusa hylid tree frogs which reveal broad spectrum antimicrobial activities. These peptides are small, and the primary structure consists of only 19-21 amino acid residues. In addition, the highly conserved residues show an amidated C-terminal with a cationic amphiphilic structure. The second structure of which is an α-helical domain [5]. A large quantity of studies have present that antimicrobial peptides have broad spectrum of antimicrobial activities. This may be related to antibacterial mechanism of peptides [6]. The main mechanism is that the cationic antimicrobial peptides physically interact with the cell membrane of the bacteria. As a result, the cell membrane tend to perforate and the cellular osmotic gradient is disrupted. The cytoplasm spill over and eventually issuing in cell lysis [7].Since the mechanism of the antimicrobial peptides differs from small molecule antibiotics, antimicrobial peptides have an extensive development and application prospect in clinical medicine. Through applying the antimicrobial peptides, people might deal with the problems of bacterial resistance. The key application of antimicrobial peptides in the future is probably to be used as anti-biofilm peptides. Microbial biofilms are produced by bacteria themselves. They gather together and adhere to the surface of bacteria. Biofilms possess high drug resistance because they are aggregates of a variety of bacteria [8].Many studies have recently proved that synthetic peptides could selectively kill bacteria in biofilms [9]. Moreover, antimicrobial peptides display the characteristics of small molecular mass, no immunogenicity, fine heat stability and so on. Therefore, they are expected to be a new class of highly effective antimicrobial agents or adjuvant drugs.Nevertheless, in order to extensively apply antimicrobial peptides to treat diseases, several obstacles and shortcomings need to be overcome. First of all, the contents of natural antimicrobial peptides are too small which makes it difficult to extract efficient peptides. Though genetic engineering and chemical synthesis methods could be used for obtaining antimicrobial peptides, it would probably make the costs increase a lot. Hence, the task of improving production efficiency and reducing costs has become the goal of future discovery and development. Secondly, the bioavailability of antimicrobial peptides is low. They are susceptible to degraded by enzymes in the acidic stomach and gastrointestinal tract through oral delivery. On top of that, after the antimicrobial peptides are absorbed, most of their active ingredients would be metabolized by the liver [10]. Therefore, modifying the structures of antimicrobial peptides to improve their microbial activity, stability and bioavailability is what researchers need to study in the future investigation.In this study, we discuss the chemical synthesis and bioactivity evaluation of a novel peptide from the Phylloseptins family. Firstly, we use the solid phase peptide synthesis method to synthesize the peptide. MS/MS fragmentation sequencing is applied to confirm the predicted primary structure. Following the synthesis of peptides, the antimicrobial effect is detected by the minimum inhibitory concentration(MIC) and the minimum bactericidal concentration(MBC) experiments. References1. Wang G, Mishra B, Lau K et al.Antimicrobial Peptides in 2014. Vanden Eynde JJ, ed. Pharmaceuticals. 2015; 8(1):123-150. 2. Diamond G, Beckloff N, Weinberg A, et al. The roles of antimicrobial peptides in innate host defense. Current Pharmaceutical Design. 2009; 15(21):2377-2392.3. Zhang R, Zhou M, Wang L, et al. Phylloseptin-1 (PSN-1) from Phyllomedusa sauvagei skin secretion: a novel broad-spectrum antimicrobial peptide with antibiofilm activity. Molecular Immunology. 2010; 47(1112):2030-20374. Frost, D.R. Amphibian Species of the World: An Online Reference. Available online: http://research.amnh.org/herpetology/amphibia/index.html.5. Leite JR, Silva LP, Rodrigues MI, et al. Phylloseptins: A novel class of anti-bacterial and anti-protozoanpeptides from the Phyllomedusa genus. Peptides. 2005; 26, 565573.6. Brogden KA. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Reviews Microbiology. 2005; 3:238-50.7. Chan DI, Prenner EJ, Vogel HJ. Tryptophan- and arginine-rich antimicrobial peptides: structures and mechanisms of action. Biochimica Et Biophysica Acta. 2006; 1758(9):1184-1202.8. Hallstoodley L, Stoodley P, Kathju S, et al. Towards diagnostic guidelines for biofilm-associated infections. Fems Immunology 65(2):127.9. Taylor PK, Yeung ATY, Hancock REW. Antibiotic resistance in Pseudomonas aeruginosa biofilms: Towards the development of novel anti-biofilm therapies. Journal of Biotechnology. 2014; 191(7):121-13010. Bruno BJ, Miller GD, Lim CS. Basics and recent advances in peptide and protein drug delivery. Therapeutic Delivery. 2013; 4, 14431467.
3. 设计方案和技术路线
一、 合成多肽1. 去保护：用哌啶去除第一个氨基酸的氨基端保护基团Fmoc。
2. 激活：用活化剂活化下一个氨基酸的羧基端。
3. 交联：活化的单体与游离的氨基反应交联，形成肽键。
4. 工作计划
2022.3.62022.3.22阅读文献，查阅相关资料，初步拟定实验方案2022.3.222022.3.31开展实验，就实验问题进行初期讨论2022.3.222022.4.14完成实验方法学和结果提交，进行中期讨论2022.4.142022.4.26整理数据，撰写毕业论文，进行最终讨论2022.4.262022.5.15提交论文终稿，做好毕业答辩准备
5. 难点与创新点
本实验采用固相多肽合成的方法合成所需抗菌肽，并能够合成在细菌中难以表现的自然多肽、加入非自然胺基酸、修饰多肽、蛋白骨架并合成拥有D构型的蛋白。
同时，运用自动多肽合成仪操作简单，大大简化了后处理操作的步骤，可以方便高效的获得大量所需多肽，并且具有较高的纯度和产率，因此具有很高的运用前景。
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