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A Novel Dermaseptin Peptide from the Defensive Skin Secretion of Phyllomedusa Camba

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A Novel Dermaseptin Peptide from the Defensive Skin Secretion of Phyllomedusa Camba MASTER OF PHILOSOPHY A novel dermaseptin peptide from the defensive skin secretion of Phyllomedusa camba Zhu, Haohao Award date: 2017 Awarding institution: Queen's University Belfast Link to publication Terms of use All those accessing thesis content in Queen’s University Belfast Research Portal are subject to the following terms and conditions of use • Copyright is subject to the Copyright, Designs and Patent Act 1988, or as modified by any successor legislation • Copyright and moral rights for thesis content are retained by the author and/or other copyright owners • A copy of a thesis may be downloaded for personal non-commercial research/study without the need for permission or charge • Distribution or reproduction of thesis content in any format is not permitted without the permission of the copyright holder • When citing this work, full bibliographic details should be supplied, including the author, title, awarding institution and date of thesis Take down policy A thesis can be removed from the Research Portal if there has been a breach of copyright, or a similarly robust reason. If you believe this document breaches copyright, or there is sufficient cause to take down, please contact us, citing details. Email: [email protected] Supplementary materials Where possible, we endeavour to provide supplementary materials to theses. This may include video, audio and other types of files. We endeavour to capture all content and upload as part of the Pure record for each thesis. Note, it may not be possible in all instances to convert analogue formats to usable digital formats for some supplementary materials. We exercise best efforts on our behalf and, in such instances, encourage the individual to consult the physical thesis for further information. Download date: 25. Sep. 2021 A novel dermaseptin peptide from the de- fensive skin secretion of Phyllomedusa camba Haohao Zhu B.Sc School of Pharmacy Faculty of Medicine, Health and Life Science Queen’s University Belfast A thesis submitted to Queen’s University Belfast for the degree of Master of Philosophy (MPhil) 2017 Contents Acknowledgements .................................................................................................................. V Declaration .............................................................................................................................. VI Abstract .................................................................................................................................. VII Chapter 1 ...................................................................................................................................1 Introduction ..............................................................................................................................1 1.1 Peptides of Phyllomedusa skin secretions ................................................................4 1.2 Antimicrobial peptides (AMPs) from the skins of Phyllomedusa .........................7 1.3.1 Dermaseptin .......................................................................................................8 1.3.2 Dermatoxin ...................................................................................................... 12 1.3.3 Distinctin.......................................................................................................... 14 1.3.4 Phylloseptin ..................................................................................................... 15 1.3.5 Phylloxin .......................................................................................................... 18 1.3.6 Plasticin............................................................................................................ 19 1.3.7 Skin polypeptide YY ....................................................................................... 21 1.3 Aims and objectives of this research..................................................................... 23 Chapter 2 ................................................................................................................................ 24 Materials and Methods ......................................................................................................... 24 2.1 Specimen biodata and secretion harvesting ......................................................... 25 2.2 Molecular cloning ................................................................................................... 25 2.2.1 mRNA isolation ............................................................................................... 25 2.2.2 cDNA library construction............................................................................. 27 2.2.3 Rapid amplification of cDNA ends-Polymerase chain reaction (RACE PCR) .......................................................................................................................... 30 2.2.4 Agarose gel electrophoresis ............................................................................ 32 2.2.5 PCR product purification .............................................................................. 33 2.2.6 Ligation ............................................................................................................ 35 2.2.7 Transformation ............................................................................................... 36 2.2.8 Blue and white colony screening ................................................................... 38 2.2.9 Isolation of recombinant DNA by cloning PCR ........................................... 38 2.2.10 Agarose gel electrophoresis analysis ............................................................. 41 II 2.2.11 Purification of selected PCR product ........................................................... 41 2.2.12 DNA sequencing reaction ............................................................................... 41 2.2.13 Purification of extension products ................................................................ 43 2.2.14 Sequencing ....................................................................................................... 43 2.3 Solid phase peptide synthesis ................................................................................ 44 2.3.1 Peptide synthesis ............................................................................................. 45 2.3.2 Peptide cleavage and rotary evaporation ..................................................... 46 2.3.3 Peptide washing .............................................................................................. 46 2.3.4 Peptide lyophilisation ..................................................................................... 47 2.3.5 Peptide yield calculation................................................................................. 47 2.4 Reversed-phase high performance liquid chromatography (HPLC) ................ 47 2.5 Peptide analysis by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry ............................................................................................................. 48 2.6 Antimicrobial assays .............................................................................................. 48 2.6.1 Microorganism inoculation ............................................................................ 49 2.6.2 Peptide preparation ........................................................................................ 49 2.6.3 Subculture ....................................................................................................... 49 2.6.4 Minimum inhibitory concentration (MIC) measurements ......................... 50 2.6.5 Viable cell counts ............................................................................................ 51 2.6.6 Minimum bactericidal concentration (MBC) measurements ..................... 51 2.7 Haemolysis assay .................................................................................................... 51 2.7.1 Peptide and control preparation ................................................................... 51 2.7.2 Horse blood preparation ................................................................................ 52 2.7.3 Haemolysis assay ............................................................................................. 52 Chapter 3 ................................................................................................................................ 54 Results ..................................................................................................................................... 54 3.1 DNA sequencing and biodata analysis.................................................................. 55 3.2 Synthesis and purification of the novel peptide ................................................... 56 3.3 Antimicrobial assays .............................................................................................. 57 3.4 Haemolysis assays ................................................................................................... 59 Chapter 4 ................................................................................................................................ 61 III Discussion ............................................................................................................................... 61 Chapter 5 ................................................................................................................................ 71 References
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