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Title: Elucidating the function of unknown proteins in the sheep tick Ixodes ricinus : a case study on a novel antibacterial peptide
Author: Burdin, Marion J.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2009
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This thesis reports on an unknown/orphan gene from the sheep tick Ixodes ricinus, so named RicOrph17, that was identified by mass spectrometry while attempting to isolate a hydrolase protein by a chromatographic approach.  RicOrph17 was “deorphanised” employing bioinformatics, gene-knockdown and recombinant protein production to determine its function. Primers were designed to screen an I. ricinus cDNA library to obtain the full length sequence of the gene.  The RicOrph17 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 RicOrph17 protein shared some structural features of the three finger protein family, including a cysteine skeleton responsible for the formation of disulfide bonds.  RicOrph17 has close homologues in many different insects species. RT-PCR analysis showed that RicOrph17 was expressed in various tissues and at different life stages. RicOrph17 was not up regulated upon bacterial challenge.  Injection of RicOrph17-dsRNA into unfed adult ticks silenced the target gene expression in the whole tick.  Such dsRNA-injected ticks challenged with E. coli showed higher morality compared to the control injected ticks.  Recombinant RicOrph17 lacking the signal peptide and the GPI-anchor peptide was produced in E. coli Origami cells to assist the determination of its function.  RecRicOrph17 possessed potent antibacterial activity against Gram +ve, -ve bacteria and yeast. FITC-labelled recRicOrph17 accumulated inside bacteria and did not remain embedded in the bacterial membrane.  Further, recRicOrph17 was demonstrated to bind DNA in gel retardation studies.  Taken together, these results suggest that RicOrph17 is a novel GPI-linked antimicrobial peptide with an intracellular target involved in tick immunity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Sheep-tick