1. 研究目的与意义
Comment: Chemical synthesis of a new sequence of peptide (KKFVPLKKIANIL-NH2) and the research of its antimicrobial activity. Meaning: Antimicrobial peptides (AMPs), concluding peptides produced by all known species, like bacteria, fungi, plants, insects, frogs and artificially-engineered mimics of antimicrobial peptides such as SNAPPs. AMPs have rapid and broad-spectrum antimicrobial activity, thus AMPs are considered as the most potential alternatives to antibiotics. Compared with antibiotics, AMPs are excellent candidate agents that subvert a number of microbial antibiotic resistance. Unlike the majority of conventional antibiotics who achieve their antimicrobial activity by disrupting various key cellular processes, AMPs target the lipopolysaccharide layers of cell membrane. The antibacterial mechanisms of some peptides are even multiple. Based on these advantages, its necessary to discover and improve their antimicrobial activity through reasonable modification of peptides sequences.
2. 文献综述
PEPTIDE-ANTIMICROBIAL AGENTS
LIN QIUYI
Nanjing University of Chinese Medicine
Antimicrobial peptides (AMPs) are widely considered as the most potential alternatives to antibiotics based on these following features. Firstly, AMPs target the lipopolysaccharide layers of cell membrane, which widely persists in microorganisms. When antibiotics target specific cellular activities, AMPs have wider and faster killing effect compared with antibiotics. Drug resistance is a problem to be solved caused by the abuse of antibiotics. New research has confirmed that antibiotic-resistant bacteria show widespread collateral sensitivity to AMPs, providing a possible way to solve drug resistance (Lzr et al., 2018). Peptides containapproximately 50 or fewer amino acids, making it easier to produce.
FEATURES OF AMPS
Most AMPs can be characterized as one of the following four types based on their secondary structures: β -sheet, α -helix, extended and loop. The αhelix AMPs are the most intensely studied AMPs, such as protegrin, magainin, cyclic indolicin. Most AMPs target bacterial cell membranes and cause disintegration of the lipid bilayer structure. The membrane-active AMPs are amphipathic with cationic and hydrophobic faces (Bahar and Ren, 2013). Cationic part ensures the initial electrostatic interaction with the anionic cell membrane and the hydrophobic part inserts the AMP molecule into membrane interior (Madani et al., 2011). Some AMPs inhibit some important pathways inside the cell such as DNA replication and protein synthesis. The structure of AMPs is crucial for their antimicrobial activities as well as the length, the net charge, hydrophobicity. The activity of AMPs base on their amphipathic structures with hydrophobic and hydrophilic faces which need at least 7-8 amino acids to compose. This doesnt mean the longer AMPs have a better biological activity. With the increase of peptide length, the cell toxicity will increase at the same time. The net charge of AMPs is the main factor for initial interaction with anionic cell membrane. The number of basic amino acids like H, K, R, should be taken into consideration. Hydrophobicity has been shown to influence the activity and selectivity of AMPs (Wang et al., 2019). Nevertheless, excessive hydrophobicity might increase the cell toxicity. Therefore, a balance between length, charge, hydrophobicity and activity is needed.
METHOD OF PEPTIDE SYNTHESIS
The most widely used method to synthesize peptide in lab is solid-phase peptide synthesis (SPPS). SPPS is a method in which molecules are covalently bound on a solid support material and synthesized step by step in a single reaction vessel using selective protecting group chemistry to protect the active side chain and NH2. SPPS applys for synthesizing peptides containing less than 70 amino acids.
MECHANISM OF AMPS
In the classical models of membrane disruption, the peptides lying on the membrane reach a threshold concentration and insert themselves across the membrane to form either peptide-lined pores in the barrel-stave model, to solubilize the membrane into micellar structures in the carpet model, or to form peptide-and-lipidlined pores in the toroidal pore model.
Table 1.Different modes of action that have been described for AMPs
Major target | Example peptides | Reference |
External proteins | Magainin 2 | (Brogden, 2005) |
Outer surface lipids | Cecropin P1 | (Strauss et al., 2010) |
Inner membrane | Melittin; daptomycin | (Brogden, 2005) |
Integral membrane proteins | Clavanin A | (van Kan et al., 2002) |
Nucleic acids | Buforin 2, | (Brogden, 2005) |
Intracellular proteins | Apidaecin | (Brogden, 2005) |
CONCLUSION
In conclusion, researches on AMPs have achieved great advances. A better understanding on their structural features and modes of action has been achieved. Though, there still remains challenges on AMPs such as: low specificity, potential toxicity, lack of guideline for rational design, AMPs still have a bright future.
REFERENCES
Lzr, V., Martins, A., Spohn, R., Daruka, L., Grzal, G., Fekete, G., Szmel, M., Jangir, P., Kintses, B., Csrg, B., Nyerges, ., Gyrkei, ., Kincses, A., Dr, A., Walter, F., Deli, M., Urbn, E., Hegeds, Z., Olajos, G., Mhi, O., Blint, B., Nagy, I., Martinek, T., Papp, B. and Pl, C. (2019).Antibiotic-resistant bacteria show widespread collateral sensitivity to antimicrobial peptides. Nature Microbiologyvolume 2018, 3, 718731.
Bahar, A. and Ren, D. (2013).Antimicrobial Peptides.Pharmaceuticals(Basel), 2013,6(12), 1543-1547.doi: 10.3390/ph6121543
Madani, F., Lindberg, S., Langel, ., Futaki, S. and Grslund, A. (2011).Mechanisms of Cellular Uptake of Cell-Penetrating Peptides. J. Biophys. 2011, 2011, 414729.
Wang, J., Song, J., Yang, Z., He, S., Yang, Y., Feng, X., Dou, X. and Shan, A. .Antimicrobial Peptides with High Proteolytic Resistance for Combating Gram-Negative Bacteria. J.Med Chem, 2019, 62(5), 2286-2304. doi: 10.1021/acs.jmedchem.8b01348
Brogden, K. (2005). Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?. Nature Reviews Microbiology, 3(3), pp.238-250.
Strauss, J., Kadilak, A., Cronin, C., Mello, C. and Camesano, T. (2010). Binding, inactivation, and adhesion forces between antimicrobial peptide cecropin P1 and pathogenic E. coli. Colloids and Surfaces B: Biointerfaces, 75(1), pp.156-164.
van Kan, E., Demel, R., Breukink, E., van der Bent, A. and de Kruijff, B. (2002). Clavanin Permeabilizes Target Membranes via Two Distinctly Different pH-Dependent Mechanisms. Biochemistry, 41(24), pp.7529-7539.
3. 设计方案和技术路线
There are three parts of the research including solid-phase peptide synthesis (SPPS), High Performance Liquid Chromatography- Mass Spectrometry (HPLC-MS) to identify the primary structure and antibacterial experiment to confirm the function of the peptide. Firstly, SPPS is a method in which molecules are covalently bound on a solid support material and synthesized step by step in a single reaction vessel using selective protecting group chemistry to protect the active side chain and NH2. To ensure the reaction going forward, the reactants are excessive and HBTU is functioned as catalyst. Secondly, HPLC is used to separate and purify the peptides synthesized above. Finally, through the relevant antibacterial experiments, the function of the peptide was identified and screened. The antibacterial effects of the polypeptide on E. coli (G-), S. aureus (G ), and C. albicans (fungus) will be observed and their minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) will be determined. Technical route: Review literature---Synthetic peptides---Sequence identification---Functional screening
4. 工作计划
TIME CONTENT PROGRESS RATE 2022.03.03~03.08 Read articles regarding to the experiment and do the presentation completed 2022.03.09~03.23 Do the experiment completed 2022.03.24~04.04 Read relative articles and write the initiating report and the review paper completed 2022.04.05~04.15 Finish the experiment To be completed 2022.04.16~05.15 Analyze the experimental data and write the research paper To be completed
5. 难点与创新点
Proteins and peptides are fundamental components of cells that carry out important biological functions. The basic distinguishing factors between peptides and proteins are size and structure. Peptides are made up of smaller chains of amino acids than proteins and tend to be less well defined in structure than proteins, which can adopt complex conformations known as secondary, tertiary, and quaternary structures. Peptides are considered as the next big thing in medical research due to their safety and stability. Whats more, their length allows them to be chemically synthesized. Antibodies are the most promising application of peptides. Compared with antibiotics, AMPs have inhibitory effect to drug resistance. This peptide (KKFVPLKKIANIL-NH2) was originally discovered from frog skin which is the source of a major part of antimicrobial peptides and has been modified to get a better antibacterial effect with low cell toxicity.
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