African horsesickness is caused by the AHSV, a member of the genus Orbivirus, family Reoviridae. Nine serotypes have been identified. The viral genome consists of ten double stranded (ds) RNA segments encoding at least 7 structural and 4 non¬structural proteins. The major core proteins VP3 and VP7 together with the minor core proteins VP1, VP4 and VP6 form the core particle surrounding the 10 dsRNA segments. An outer capsid, consisting of two major structural proteins VP2 and VP5 surrounds the core. VP2 is the most variable of the proteins within the AHSV serogroup and carries serotype specific epitopes which induce a protective immune response against virulent homologous AHSV challenge. The VP2 protein is therefore the antigen of choice for the development of a subunit vaccine against AHSV. It has been shown that protection against AHSV-4 can be achieved by vaccination with AHSV VP2 protein. The AHSV-3 VP2 protein has previously been cloned and expressed as baculovirus recombinant protein in our laboratory. The recombinant protein induced only a weak neutralising immune response. It has been determined in this investigation that the majority of recombinant AHSV-3 VP2 proteins expressed in Sf-9 insect cells are in an insoluble, aggregated form. This is likely to be the cause of the poor neutralising immune response induced by this protein. In order to investigate this problem two strategies were adopted. First an attempt was made to chemically solubilise the particulate VP2 protein and refold the protein into a form that may present the neutralising epitopes more appropriately. The solubilisation of the protein with 6M Guanidinium HCI was successful, but the largest percentage of the protein was again rendered insoluble during the refolding process which involves the removal of Guanidinium HCI by column chromatography. The chemical solubilisation therefore proved to be too inefficient to provide a solution to the problem. The second method for increasing the solubility and immunogenicity of the VP2 protein was by co-expression of VP2 and VP5, the two outer capsid proteins of AHSV¬3. For the dual expression of the two proteins it was necessary to characterise the AHSV-3 VP5 gene and express it as a baculovirus recombinant first. The VP5 gene was therefore sequenced. A nucleotide sequence of 1566 bp was determined encoding a peptide of 505 amino acids with a predicted size of 56K. The VP5 was expressed as baculovirus recombinant using the baculovirus Bac-to-Bac™ expression system. The yield of VP5 was low but was nevertheless better than the expression levels of AHSV-9 VP5 gene using an alternative baculovirus expression system. AHSV-3 VP2 and VP5, were cloned respectively under the polyhedrin and p10 promoters of the pFastbac dual transfer vector of the Bac-to-Bac™ baculovirus expression system. mRNA transcription of both AHSV-3 VP2 and VP5 genes in Sf-9 cells was shown. The expression of VP2 was also demonstrated but VP5 was very poorly expressed by the dual recombinant. Further research to determine the effect co-expression of AHSV-3 VP5 in the AHSV-3 VP2 antigenicity is needed. / Dissertation (MSc Agric (Genetics))--University of Pretoria, 2006. / Genetics / unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/30189 |
| Date | 07 December 2006 |
| Creators | Filter, Renate Dorothea |
| Contributors | Prof H Huismans, upetd@up.ac.za |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2000, 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. |
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