Title page for ETD etd-06232005-171252


Document Type Master's Dissertation
Author Meyer, Quinton Christian
URN etd-06232005-171252
Document Title The presentation of an HIV-1 neutralizing epitope on the surface of major structural protein VP7 of African horse sickness virus
Degree MSc (Genetics)
Department Genetics
Supervisor
Advisor Name Title
Prof H Huismans Committee Chair
Keywords
  • African horse sickness virus research
Date 2003-04-01
Availability unrestricted
Abstract
Two particulate structures based on AHSV major structural protein VP7, are under investigation as possible vectors for epitope display. Due to the extreme insolubility of the VP7 protein it aggregates into large hexagonal crystalline particles when expressed in an insect cell expression system (Chuma et aI, 1992). To investigate its ability to present immunologically important epitopes to the immune system, VP7 mutants have been constructed that allow the presentation of foreign peptides on the surface these particles (Maree, 2000).

In part of this study AHSV core-like particles resulting from the co-expression of the two major structural proteins VP3 and VP7 were under investigation. Due to the low solubility of VP7, low core-like particle yields are obtained during co-expression (Maree, 2000). As a result not enough of the particles can be produced to make its use viable. To increase the core-like particle yield it is necessary to increase the solubility of VP7. Several amino acids have been implicated in the observed low solubility of VP7 (Monastyrskaya et aI, 1997). One of these amino acids was targeted for site-specific mutation in an effort to increase protein solubility. Leucine 345 is located on the ninth C-terminal helix in the bottom domain of VP7 and was substituted to arginine, a polar positively charged residue. The substitution was made in wild type VP7 as well as in insertion mutants 177 and 200. The mutation was effected via PCR and the resulting mutant genes were expressed in the Bac-To-Bac expression system. The newly constructed mutant proteins were further investigated for an increase in solubility by differential and sucrose density centrifugation.

The site specific mutation in wild type VP7 and insertion mutant 200 resulted in a slight increase in solubility whereas the mutation in mutant 177 had no effect on solubility. As a result of failing to significantly increase the solubility of these mutants, no increase in the core-like particle yield was achieved.

In the second part of this study recombinant VP7 crystals were constructed which present a single and triple repeat of the HIV-1 transmembrane protein gp41 neutralizing antibody epitope, ELDKWA. To this end, oligonucleic acid adaptors coding for the epitope repeats were designed and cloned into VP7 insertion sites 144 and 177. The newly constructed VP7 mutant genes were expressed in the Bac-to-Bac expression system and the recombinant proteins were investigated for its solubility and crystal formation by sucrose density gradient centrifugation. All of the epitope presenting constructs were significantly less soluble than insertion mutants 144 and 177 and retained its ability to assemble into large hexagonal crystals. Gradient purified particles were injected into Balb/c mice and the epitope specific antibody response determined by dot and western blot analysis. Although a humoral immune response was induced against the VP7 constructs none were able to induce antibody responses specific to the ELDKWA epitope.

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:

Meyer, QC 2002, The presentation of an HIV-1 neutralizing epitope on the surface of major structural protein VP7 of African horse sickness virus, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-06232005-171252/ >

H993/ag

Files
  Filename       Size       Approximate Download Time (Hours:Minutes:Seconds) 
 
 28.8 Modem   56K Modem   ISDN (64 Kb)   ISDN (128 Kb)   Higher-speed Access 
  dissertation.pdf 3.65 Mb 00:16:52 00:08:40 00:07:35 00:03:47 00:00:19

Browse All Available ETDs by ( Author | Department )

If you have more questions or technical problems, please Contact UPeTD.