1. 研究目的与意义
Content:Evaluation of antimicrobial activity:①Minimal inhibitory concentration(MIC) assay: the antimicrobial activity of two dermaseptins was assessed by determining their MICs against reference strains of a Gram-positive bacterium, Staphylococcus aureus , a Gram-negative bacterium , Escherichia coli, and a yeast, Candida albicans .②Minimum bactericidal (MBC) assayEvaluation of anticancer activity: use the MTT cell viability assay to evaluate the growth modulating effects of two dermaseptins from the skin secretion of the Mexican leaf frog on human cancer cells such as the human breast carcinoma cell Line, MDA-MB-435s; the non-tumorigenic mammary gland cell line, MCF-7 and U251MG, the human neuronal glioblastoma (Astrocytoma).Meaning：As we all known the amphibian skin may be regarded as an enormous storehouse of biogenic amines and active polypeptides and 88% of the total of 7302 amphibian species being anurans (frogs and toads). Antimicrobial peptides have a smaller molecular weight, good heat resistance, smaller antigenicity and broad antimicrobial activity. Some antimicrobial peptides also have anti-viral, anti-fungal and inhibit cancer cell growth. We hope through research to discover novel peptide and an alternative to traditional surgery antineoplastic chemotherapy, as well as easy to produce drug resistance of new antimicrobial agents. This experiment was designed to identify and synthesize new antimicrobial and anticancer peptides.
2. 文献综述
Antimicrobial Peptides: New Insight for Antibiotic Drug DiscoveryZUO Wen-wen(School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210046, China)Abstract: Recently, the infection disease served as a serious threat to human health, although some antibiotics have made great achievement in the clinical treatment of infection diseases. However, with the abuse of antibiotics in various fields, drug residues, bacterial resistance problem and other issues have become increasingly prominent and caused widespread concern in the field of global public health. Now, an increasing number of countries and regions begin to regulate the use of antibiotics and other drugs strictly to reduce the incidence of this series problems. The emergence and spread of the "super bacteria" has made a warning to humans. Thus, the development of new anti-infective drugs is becoming increasingly important. Antimicrobial Peptides (AMPs) is a class of small peptides with biological activity which encoded by specific genes base of human body and inducted by the outside world and they are key components of the ancient innate immune system and very important part of natural organism vivo defense system. Antimicrobial peptides can fight bacteria, fungi, viruses and even cancer cells, with characteristics of small molecular weight, good water solubility, broad-spectrum activity and low immunogenicity. Based on the above characteristics, antibacterial peptides will have broad application prospects in medicine, agriculture, food industry and other fields. The review will give a brief introduction of classification, biological activity, mechanism of action and application status of antimicrobial peptides. Key words: antimicrobial peptides; activity; mechanism; application摘要：近年来，感染性疾病任严重地威胁着人类的健康，虽然抗生素在其临床治疗中带来很大成效，但随着抗生素等药物在各领域的滥用，药物残留、细菌耐药性等问题日益突出，引起了全球公共健康领域的广泛关注，越来越多的国家和地区开始严格规范抗生素等药物的使用。
超级细菌的出现和蔓延，给人类敲醒了警钟，因此新型抗感染药物的开发日趋显得重要。
抗菌肽（AMPs）是由机体特定基因编码并经由外界诱导产生的一类具有生物活性的小分子多肽，是自然界生物体内非常重要的防御体系。
3. 设计方案和技术路线
Research programs:1.Peptide synthesisEach natural antimicrobial peptide and their respective cationicity -enhanced analogues, were separately synthesised by solid phase methodology using Rink amide resin and standard Fmoc chemistry, by means of an automated peptide synthesiser (Protein Technologies, USA), followed by standardised deprotection and cleavage from resin. Each synthetic peptide was analysed by reverse phase HPLC and MALDI-TOF mass spectrometry to establish both degree of purity and authenticity of structure.2.Minimum inhibitory concentration (MIC) assayAntimicrobial assay was carried out to detect the minimum inhibitory concentration of each peptide on the microbes S.aureus, E coli and C.albicans. Three replicates should be performed for each peptide on those three microorganisms.3.Minimum bactericidal concentration (MBC)assayThe culture containing different concentration of peptides should be spotted on Mueller-Hinton agar (MHA) plate and the plate should be subsequently incubated for 24 hours for measurement of minimum bactericidal concentrations (MBCs) which were defined as the concentration of peptide from the MIC experiments from which no colonies could be grown.4.Culture and maintenance of human cancer cell linesThe human breast carcinoma cell Line, MDA-MB-435s; the non-tumorigenic mammary gland cell line, MCF-7 and U251MG and the human neuronal glioblastoma (Astrocytoma) has been carried out for assessment of growth modulating effects of peptides on human cancer cells using the MTT cell viability assay技术路线：见开题报告附件
4. 工作计划
TIME CONTENT PROGRESS RATE2022.03.03-03.08 Read articlesRegarding to the experiment and do the presentation completed 2022.03.09-03.23 Do the experiment completed2022.03.24-04.04 Read relative articles and write the initiating report and the review completed2022.04.05-04.15 Finish the experiment To be completed2022.04.16-05.15 Analyze the experiment data and write the research paper To be completed
5. 难点与创新点
We use frog skin secretions to isolate and extract peptides.At the time of Erspamers early research with frog skins the analytical tool kit was rather primitive and required the skinning of from several hundred individuals Besides, it was very time-consuming to determine the structure of the skin compounds. Today the conditions have entirely changed in a dramatic way: the skin secretion obtained by mild electrical stimulation of a single frog yield in sufficient material for analysis in a couple of days and leaves the animal unharmed; an invaluable ethical aspect in times of the global amphibian decline.
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