1. 研究目的与意义
内容：研究抗菌肽QUB-1985 GLLSGILGVGKKIVCGLSGLC对于白色念珠菌，大肠杆菌，金黄色葡萄球菌的抗菌活性。
意义：在抗生素遭到滥用的情况下，细菌及病毒的耐药性日益提高，抗菌肽作为一种选择性高，抗微生物活性高，易于提取的化学物质，是相当有前景的新型抗生素，研究其抗菌活性对于临床治疗新药研发都有积极意义。
2. 文献综述
Although a variety of therapeutic agents have been discovered to treat diseases caused by bacteria, microbial drug resistance is a raising problem which needs scientists to deal with. With the evolution of microorganisms, common antibiotics could hardly kill all kinds of bacteria and viruses. Even some of them would do harm to normal human cells and immunity. There are large distinctions among microbial cells and human cells, which means drugs with selective antimicrobial activity may be more effective and efficient. From some previous researches, antimicrobial peptides are considered promising as a novel therapeutic (Mangoni, Maisetta et al. 2008). In fact, amphibian skin proves to be a rich source of antimicrobial peptides.The habitats of different amphibians could be cosmopolitan. Extending from inside the Arctic Circle in the north to as far south as southern Chile and the Patagonian grasslands of southern Argentina, the ranid frog may live in various living conditions. The ranid frog show apparent preference for humid aquatic and terrestrial environments, which are normally rich for pathogens. However, unless the skin surface is broken, frogs are rarely infected by microorganisms. Thus, it is obvious that these amphibians have a complete anti-microbial and anti-fungal mechanism on their skin surface (Clarke 1997). During the millions of years evolution and improvement, they have achieved the ability to defend themselves by the skin secretions. Skin, which plays the lead role in defending frogs, has three main functional parts: mucus glands, granular glands and alveolar glands (Savelyeva, Ghavami et al. 2014).Granular glands are basically found over the dorsal area of the ranid frogs and one of their functions is to synthesize and store antimicrobial peptides. Once the host is under attack or even touched slightly, these peptides may be immediately secreted into mucus to protect the organisms (Rollins-Smith 2009). Firstly, α-adrenergic agonists may lead to the contraction of the serous cells. Then the cells may discharge not only the antimicrobial peptides but also their cytosolic components and genetic material (Holmes and Balls 1978). The mucus from frogs is the main source of antimicrobial peptides, which means we can get access to the research materials without killing or hurting, just by slight touch or current stimulation.There are many distinctions between microbial and mammalian cells, such as membrane composition and architecture, energetics like transmembrane potential and polarization, and structural features including sterols, lipopolysaccharide and peptidoglycan, which may indicate targets to antimicrobial peptides. These messages may be translated to multifarious selective toxicity within peptides, so that peptides may achieve antimicrobial bioactivity to selectively damage distinct bacteria without doing any harm to host cells (Matsuzaki 2009). This reaction is based on the biological background of interaction and the properties of bacteria and peptides (Yeaman and Yount 2003). Antimicrobial peptides are also the largest group among lytic peptides (Shai 1999). The major target of antimicrobial peptides is bacterium membranes. The structures of α-helix and β-sheet may contribute to the properties of membrane permeation and damage. The antimicrobial activity usually occurs at the shift of cell membrane hydrophobichydrophilic seal. There are two major mechanisms to explain the permeation or lysis of membranes (Ambroggio, Separovic et al. 2005). The first is the barrel-stave or pore-forming mechanism, where α-helical amphipathic peptides bind initially to the outside of the lipid bilayer, and then penetrate the bilayer to produce defined pores which are oriented perpendicular to the plane of the bilayer. A minimum of amino acid residues is required to span the bilayer, but there are examples where smaller peptides can dimerize to effect full penetration of the barrier. The second process is called the carpet mechanism, where peptides remain bound to the membrane interface and disrupt the bilayer by a detergent-like or carpet-like effect. Above a critical concentration, holes are formed due to strain on the bilayer, and the membrane degrades into micelle-like complexes (Pukala, Bowie et al. 2006). During my research on the peptide QUB-1985 GLLSGILGVGKKIVCGLSGLC, my attention would be mainly focused on the antimicrobial activity toward the gram-positive bacterium, the gram-negative bacterium and thve Candida albicans. The structure of the peptide QUB-1985 GLLSGILGVGKKIVCGLSGLC has a high degree of similarity to that of brevinin family (Wang, Evaristo et al. 2010). Peptides from brevinin family shows high selective toxicity towards cancer cells and microorganisms (Kwon, Hong et al. 1998). However, the hemolytic activity of peptides is a problem remained to be solved. Although peptides may carry selective toxicity able to kill various microorganisms and defend the human body, there is possibility that they would lyse host cells (Hancock 1997). This means these peptides may do harm to human cells when used as therapeutic agents. In some previous researches, the cyclic β-sheet structure proves to be related to the balance of hemolytic and antimicrobial activities (Kondejewski, Jelokhani-Niaraki et al. 1999). Thus, more attention should be focused on studying novel peptides with high antimicrobial activity but less harm to mammalian cells (Shin, Yang et al. 2001).ReferencesAmbroggio, E. E., et al. (2005). "Direct visualization of membrane leakage induced by the antibiotic peptides: maculatin, citropin, and aurein." Biophysical journal 89(3): 1874-1881. Clarke, B. T. (1997). "The natural history of amphibian skin secretions, their normal functioning and potential medical applications." Biological Reviews 72(3): 365-379. Hancock, R. E. (1997). "Peptide antibiotics." The Lancet 349(9049): 418-422. Holmes, C. and M. Balls (1978). "In vitro studies on the control of myoepithelial cell contraction in the granular glands of Xenopus laevis skin." General and comparative endocrinology 36(2): 255-263. Kondejewski, L. H., et al. (1999). "Dissociation of antimicrobial and hemolytic activities in cyclic peptide diastereomers by systematic alterations in amphipathicity." Journal of Biological Chemistry 274(19): 13181-13192. Kwon, M.-Y., et al. (1998). "Structure-activity analysis of brevinin 1E amide, an antimicrobial peptide from Rana esculenta." Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology 1387(1): 239-248. Mangoni, M. L., et al. (2008). "Comparative analysis of the bactericidal activities of amphibian peptide analogues against multidrug-resistant nosocomial bacterial strains." Antimicrobial agents and chemotherapy 52(1): 85-91. Matsuzaki, K. (2009). "Control of cell selectivity of antimicrobial peptides." Biochimica et Biophysica Acta (BBA)-Biomembranes 1788(8): 1687-1692. Pukala, T. L., et al. (2006). "Host-defence peptides from the glandular secretions of amphibians: structure and activity." Natural product reports 23(3): 368-393. Rollins-Smith, L. A. (2009). "The role of amphibian antimicrobial peptides in protection of amphibians from pathogens linked to global amphibian declines." Biochimica et Biophysica Acta (BBA)-Biomembranes 1788(8): 1593-1599. Savelyeva, A., et al. (2014). An overview of Brevinin superfamily: structure, function and clinical perspectives. Anticancer Genes, Springer: 197-212. Shai, Y. (1999). "Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell non-selective membrane-lytic peptides." Biochimica et Biophysica Acta (BBA)-Biomembranes 1462(1): 55-70. Shin, S., et al. (2001). "Antibacterial, antitumor and hemolytic activities of α‐helical antibiotic peptide, P18 and its analogs." The Journal of Peptide Research 58(6): 504-514. Wang, L., et al. (2010). "Nigrocin‐2 peptides from Chinese Odorrana frogsintegration of UPLC/MS/MS with molecular cloning in amphibian skin peptidome analysis." FEBS journal 277(6): 1519-1531. Yeaman, M. R. and N. Y. Yount (2003). "Mechanisms of antimicrobial peptide action and resistance." Pharmacological reviews 55(1): 27-55. 中文翻译：尽管人们已经研制出大量药物用于治疗微生物所导致的疾病，但是微生物耐药性也在逐渐变为一个急需科研人员关注的问题。
随着细菌和病毒的不断进化，常规的抗生素已经不能对抗所有的致病微生物了，甚至有一些抗生素对于人体正常细胞以及免疫能力都会造成伤害。
事实上，人体细胞和微生物细胞之间存在着巨大的差别，这意味着具有选择性抗菌活性的药物与现有抗生素相比将会更加快速有效。
3. 设计方案和技术路线
1、 通过固相化学合成法对肽进行合成。
2、 利用高效液相法进行纯化。
3、 通过质谱法对肽的结构进行验证。
4. 工作计划
3.08称取合成所需的氨基酸。
3.10开始合成氨基酸。
3.13利用质谱法检验肽结构。
5. 难点与创新点
固相合成反应在一简单反应器皿中便可进行；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；增加溶剂化，减少中间的产物聚焦；固相载体上肽链和轻度交联的聚合链紧密相混，彼此产生一种相互的溶剂效应，这对肽自聚集热力学不利而对反应适宜。
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