1. 研究目的与意义
青蛙（包括蟾蜍）是一种药用价值很高的经济动物，其全身是宝，蟾衣、蟾酥、干蟾、蟾头、蟾舌、蟾肝、蟾胆等均为名贵药材，应用于临床抗肿瘤、抗病毒等。
目前，来自青蛙皮肤的分泌物已被证明是抗微生物分子的不可替代的丰富来源，其中蟾蜍作为传统中药，其耳后腺及表皮腺体的分泌物（称为蟾酥），具有破症结、行水湿、化毒、杀虫、定痛的功效。
它们的表皮分泌物存在着许多生物大分子，而我们从小分子角度研究，通过青蛙皮肤分泌物中分离提取的抗菌肽，进一步证实其抗菌机理。
2. 文献综述
IntroductionThus far, people rely on antimicrobial drugs using for curing infectious diseases in excess, which contributes to the significant increase of the resistance among all kinds of microbial strains, even including the cancer cells [1]. Nowadays, what has turned into main risks of the clinical medicine is that bacteria are increasingly resistant to antibiotics [2].So it is high time that we found the therapeutic capacity of neo-molecules against multi-drug resistance and provided new approaches to address the problem that pathogenic bacteria generate more drug resistance [3]. Due to the wide distribution of antimicrobial peptides (AMPs) in nature and the particular characterizations of them, such as broad antibacterial spectrum, strong heat stability, small molecular weight, little immunogenicity and so on, AMPs show extensive activity against multiple gram-negative and gram-positive bacteria, fungi and other strains, also involving the cancer cells [1]. Despite the lower efficacy of them against the pathogens which are easier to be infected than that of some traditional antibiotics, AMPs possess unique sterilization mechanism, which can kill resistant strains by analogous concentrations. In contrast with common antibiotics, they can attack rather quickly targets on various bacterial membranes as well [4]. In addition, many studies revealed that AMPs were not hindered by the mechanism of resistance happening on antibiotics and thus pathogenic bacteria might not tend to generate drug resistance because of the interaction between AMPs and the cell membranes or various targets [1,4]. Therefore, AMPs are described as the optimum substitutions for common antibiotics, promising to be developed into a potential generation of peptides antibiotics [5]. One kind of frogs named toad is usually used as a traditional Chinese medicine. Considering that frogs are amphibians with exposed skin, which cannot effectively prevent moisture evaporation from their bodies, they have to live in the wet environment where a multitude of bacteria are easier to breed. Based on such circumstances, we could extrapolate that the skin of frogs may certainly exist a sort of substance to resist microbial attack. There are some findings recently, hence, that phylloseptins from the skin secretion of phyllomedusine frogs belong to the AMPs family [6]. The peptides of this family generally contain 19-21 amino acids and possess amidated C terminal, a cationic amphiphilic structure and a large proportional α-helical domain [5,6]. On account of the highly-conserved structure (sequence FLSLI[L]P [4]), these AMPs destroy the cell membrane to form ion channels and bacteria would eventually die because of failing to keep the normal osmotic pressure. This shows effective bacteriostatic and bactericidal activity of AMPs [1]. Furthermore, these peptides exhibit little hemolytic activity, namely, that means they exert a small effect upon red blood cells and only have low toxicity to mammalian cells under a very high concentration [4,7]. It is concluded that phylloseptins act prior on prokaryotic rather than eukaryotic cell membranes as a result [6,7]. As the high-throughput molecular technologies appear nowadays, peptides sequencing can be applied to the discovery of peptides by means of tandem mass spectrometry, cDNA cloning and pharmacological screening, which makes quick analysis of structural data and abundant libraries of peptide sequences [4]. Among these technologies, Fmoc solid-phase peptide synthesis (SPPS) is the first choice for peptide synthesis. Since the quantities of synthetic peptides rise continually into clinical tests, this method can obtain Fmoc structure modules of extremely high quality without much cost, simply compounding a mass of natural peptides [8]. Another advantage of Fmoc SPPS is that it is liable to automation for no need of corrosive Trifluoroacetic Acid (TFA) in the synthetic process and releasing strong-UV-absorptive fluorenyls in the deprotection. This, therefore, quite alleviates the difficulties of the purify of Fmoc modules in every step, improving on the purify of synthetic peptides [8]. Topical peptides research in turn helps to update and extend the applications of Fmoc SPPS [8].Here, this experimental study synthesized a peptide (sequence FLSLIPKIISAISALIKHF-NH2) via Fmoc SPPS combined with mass spectrometry to verify the sequence of it (the significance of this MS-based sequencing method for scientific research has been reported [9].)Then we identified and screened its function by relevant anti-microbial experiments, including observing its abilities against Escherichia coli, Staphylococcus aureus and Candida albicans. Finally, we could know its minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). In the future, natural AMPs might be designed or modified appropriately to improve stability and bioavailability, remove hemolytic activity and reduce preparing cost simultaneously based on the efficient capability of antimicrobial activity [5]. The appearance of pharmacodynamic and pharmacokinetic problems AMPs caused in their exploitations and clinical applications could be overcame, such as peptide aggregation, the in vivo half-life of AMPs and the needed frequency of dose [4]. So almost all modified AMPs might be more superior to natural AMPs as therapeutic agents [4]. However, the versatility and adjustability of peptides antibiotics lead to uncertain changes in the therapy [10]. Additionally, there exists a part inconsistency between anti-microbial experimental simulation and the real environment or mode where bacteria breed, which all result in great challenges with their wider range of implementation.Taken together, the skin secretions from these frogs have proved to be a plentiful and irreplaceable source of antimicrobial molecule [4]. And the underlying treatment applications of various AMPs should be paid more attention to. They can restrain multi-drug-resistant pathogenic microorganisms effectively and make causative agents not easy to generate resistance, offering instructive meanings for the treatment of infectious diseases [4]. To tackle all above-mentioned problems, the study on synthetic peptides by Fmoc SPPS may have played a vital role in bringing about new discovery of active peptides and a strategy of AMPs applying for the medical treatment, which establishes a powerful basis for research and development of drug design.References1. Lakshmaiah Narayana J, Chen JY. Antimicrobial peptides: Possible anti-infective agents. Peptides. 2015; 72: 88-94.2. Chen HL, Su PY, Shih C. Improvement of in vivo antimicrobial activity of HBcARD peptides by D-arginine replacement. Appl Microbiol Biotechnol. 2016; 100: 9125-32.3. Ashby M, Petkova A, Gani J, et al. Use of Peptide Libraries for Identification and Optimization of Novel Antimicrobial Peptides. Curr Top Med Chem. 2017; 17: 537-53.4. Azevedo Calderon L, Silva Ade A, Ciancaglini P, et al. Antimicrobial peptides from Phyllomedusa frogs: from biomolecular diversity to potential nanotechnologic medical applications. Amino Acids. 2011; 40: 29-49.5. Gao Y, Wu D, Xi X, et al. 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: 6. Wan Y, Ma C, Zhou M, et al. Phylloseptin-PBa--A Novel Broad-Spectrum Antimicrobial Peptide from the Skin Secretion of the Peruvian Purple-Sided Leaf Frog (Phyllomedusa Baltea) Which Exhibits Cancer Cell Cytotoxicity. Toxins (Basel). 2015; 7: 5182-93.7. 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. Mol Immunol. 2010; 47: 2030-7.8. Behrendt R, White P, Offer J. Advances in Fmoc solid-phase peptide synthesis. J Pept Sci. 2016; 22: 4-27.9. Yefremova Y, Al-Majdoub M, Opuni KF, et al. "De-novo" amino acid sequence elucidation of protein G'e by combined "top-down" and "bottom-up" mass spectrometry. J Am Soc Mass Spectrom. 2015; 26: 482-92.10. Rafferty J, Nagaraj H, McCloskey AP, et al. Peptide Therapeutics and the Pharmaceutical Industry: Barriers Encountered Translating from the Laboratory to Patients. Curr Med Chem. 2016; 23: 4231-59.附：目前，人们对用于治疗感染性疾病的抗菌药物的过量依赖，导致了各种微生物菌株之间以及癌细胞的抗性大大增加[1]。
如今，越来越多的抗生素抗性细菌已成为临床医学的主要威胁 [2]。
因此，现在我们迫切需要找到对多药耐药菌株具有治疗效应的新分子，为解决病原菌产生更多耐药性的问题提供新的方法途径[3]。
3. 设计方案和技术路线
设计方案：先用固相合成法，重复添加精密称定的所需氨基酸，从C端（羧基端）向N端（氨基端）合成。
为防止副反应的发生，参加反应的氨基酸的侧脸都受到Fmoc保护，C端游离并在反应前活化。
加入树脂以固定得到该条多肽FLSLIPKIISAISAISALIKHF-NH2，冻干备用。
4. 工作计划
2022年3月初-3月中旬查阅大量文献，选定研究课题，初步学习并制定实验方案；进行第一次开题报告指导会议。
2022年3月中旬-3月底精密称定氨基酸，合成多肽，冻干备用；撰写开题报告并提交审核。
2022年4月初-4月中旬完成该条多肽的序列鉴定以及功能筛选；进行第二次论文研究工作中期检查会议，提交方法学和实验结果。
5. 难点与创新点
青蛙（包括蟾蜍）是一种药用价值很高的经济动物，其全身是宝，蟾衣、蟾酥、干蟾、蟾头、蟾舌、蟾肝、蟾胆等均为名贵药材，应用于临床抗肿瘤、抗病毒等。
目前，从它身上提取的蟾酥以及蟾衣是我国紧缺的中药材。
理论分析，它们的表皮分泌物必然存在着许多生物大分子，而我们从小分子角度研究，通过青蛙皮肤分泌物中分离提取的抗菌肽，进一步证实其抗菌机理。
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