Title page for ETD etd-10072005-103940


Document Type Master's Dissertation
Author Olivier, Nicholas Abraham
URN etd-10072005-103940
Document Title Isolation and cahracterization of antibacterial peptides from hemolymph of the soft tick, Ornithodoros savignyi
Degree MSc (Biochemistry)
Department Biochemistry
Supervisor
Advisor Name Title
Prof A W H Neitz
Dr A R M D Caspar Committee Co-Chair
Keywords
  • Ornithodoros savignyi
  • ticks research
  • ticks microbiology
  • ticks as carriers of disease
  • argasidae
Date 2002-04-01
Availability unrestricted
Abstract
Invertebrates do not possess an adaptive immune system, but rely on several mechanisms similar to the innate immune system of mammals. The synthesis and release of a host of potent antimicrobial proteins is an important component of this immune response.

The antibacterial activity in the hemolymph of Ornithodoros savignyi is specific for Gram-positive bacteria, and the synthesis and release of the antibacterial factors need to be induced by challenging the ticks with heat-killed Gram-negative bacterial suspensions. The induction of the factors is very rapid, leading to a maximal response within one hour following bacterial challenge. The factors are stable at high temperatures, and were found to be protein in nature. By using reverse phase high performance liquid chromatography, four fractions exhibiting antibacterial activity were identified in the hemolymph of immune challenged ticks. Four antibacterial peptides were isolated from these fractions, and the mass analyses of the peptides indicate that there are at least two different antibacterial peptides present in the hemolymph. The N-terminal amino acid sequence of one of the peptides was determined, and the analysis showed that the peptide has high homology with defensin peptides isolated from other tick species. This led to the putative classification of the peptides as part of the invertebrate defensin family.

The presence of lysozyme in O. savignyi was studied using molecular biological methods. Vertebrate and invertebrate lysozyme sequences were used to design a lysozyme-specific primer, which was used to amplify specific DNA products from whole tick cDNA using the polymerase chain reaction (PCR). The conditions for the amplification reaction were optimized, the products of the optimized reaction were cloned into a cloning vector and the nucleotide sequences of the products were determined. The nucleotide sequences were used for similarity searches of sequence databases to determine homology with sequences of known proteins. It is deduced the degenerate primer was not specific for lysozyme and did not playa significant role in the amplification of the PCR products. This method is thus not feasible for the investigation of the lysozyme of O. savignyi.

2002 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.

Please cite as follows:

Olivier, NA 2002, Isolation and characterization of antibacterial peptides from the hemolymph of the soft tick, Ornithodoros savignyi, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-10072005-103940/ >

H1173/ag

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