Document Type Master's Dissertation Author Vreede, Frank Theodoor URN etd-08182010-122817 Document Title Cloning, characterization and expression of the gene that encodes the major neutralization-specific antigen of African horsesickness virus serotype 3 Degree MSc Department Genetics Supervisor
Advisor Name Title Prof H Huismans Supervisor Keywords
- horsesickness virus serotype 3
Date 1995-04-01 Availability unrestricted AbstractThe aim of this investigation was to clone, characterize and express the gene that encodes the outer capsid protein, VP2, of African horsesickness virus (AHSV), with a view to the evaluation of this protein as a subunit vaccine.
The VP2 gene of AHSV serotype 3 (AHSV-3) was cloned as incomplete cDNA fragments of the genome segment 2 double-stranded (ds)RNA, sequenced in its entirety and compared with previously published cognate sequences of AHSV-4, Epizootic hemorrhagic disease virus (EHDV)-l and various bluetongue virus (BTV) serotypes. AHSV-3 genome segment 2 was shown to be 3221 nucleotides in length, encoding a protein of 1057 amino acids with a 50.5% identity to AHSV-4 VP2. Two areas of high variability (approximately 65%) were identified adjacent to the conserved termini. The N-proximal region (amino acids 128-309) exhibited significant hydrophilicity, suggesting a possible role in the determination of the serotype-specific immune response. Orbivirus interserogroup comparisons of VP2 amino acid sequences revealed extreme variability, although an overall structural conservation was demonstrated.
Oligonucleotide primers derived from the AHSV-3 genome segment 2 terminal nucleotide sequences were used for PCR amplification and cloning of full length segment 2 cDNA. The cloned gene was expressed in a baculovirus expression system and the expressed VP2 protein was shown to react specifically with anti AHSV-3 serum in Western blots.
Although the yields of VP2 in the baculovirus system were low, due to a possible toxic effect on the host cells, sufficient antigen was obtained for further future investigations into the efficacy of VP2 as a possible subunit vaccine against AHSV.
© 1994 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:
Vreede, FT 1994, Cloning, characterization and expression of the gene that encodes the major neutralization-specific antigen of African horsesickness virus serotype 3, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-08182010-122817/ >
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