This thesis reports on an unknown/orphan gene from the sheep tick <i>Ixodes ricinus, </i>so named <i>RicOrph17, </i>that was identified by mass spectrometry while attempting to isolate a hydrolase protein by a chromatographic approach. <i>Ric</i>Orph17 was “deorphanised” employing bioinformatics, gene-knockdown and recombinant protein production to determine its function. Primers were designed to screen an <i>I.</i><i> ricinus </i>cDNA library to obtain the full length sequence of the gene. The <i>RicOrph17</i> open reading frame encodes a 145 amino acid polypeptide, including a putative 26-amino acids signal peptide and a 26-coding region with a putative GPI-anchoring site. The deduced <i>Ric</i>Orph17 protein shared some structural features of the three finger protein family, including a cysteine skeleton responsible for the formation of disulfide bonds. <i>Ric</i>Orph17 has close homologues in many different insects species. RT-PCR analysis showed that <i>RicOrph17</i> was expressed in various tissues and at different life stages. <i>RicOrph17</i> was not up regulated upon bacterial challenge. Injection of <i>RicOrph17-</i>dsRNA into unfed adult ticks silenced the target gene expression in the whole tick. Such dsRNA-injected ticks challenged with <i>E. coli </i>showed higher morality compared to the control injected ticks. Recombinant <i>Ric</i>Orph17 lacking the signal peptide and the GPI-anchor peptide was produced in <i>E. coli </i>Origami cells to assist the determination of its function. Rec<i>Ric</i>Orph17 possessed potent antibacterial activity against Gram +ve, -ve bacteria and yeast. FITC-labelled rec<i>Ric</i>Orph17 accumulated inside bacteria and did not remain embedded in the bacterial membrane. Further, rec<i>Ric</i>Orph17 was demonstrated to bind DNA in gel retardation studies. Taken together, these results suggest that <i>Ric</i>Orph17 is a novel GPI-linked antimicrobial peptide with an intracellular target involved in tick immunity.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:510535 |
| Date | January 2009 |
| Creators | Burdin, Marion J. |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=59604 |
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