Title page for ETD etd-08302012-103144


Document Type Master's Dissertation
Author Pillay, Priyen
Email kraven.priyen@gmail.com
URN etd-08302012-103144
Document Title Expression of the VP1 antigen from foot-and-mouth disease virus in a bacterial and plant-based expression system
Degree MSc
Department Plant Science
Supervisor
Advisor Name Title
Dr R Chikwamba Co-Supervisor
Prof K J Kunert Supervisor
Keywords
  • plant-based expression system
  • bacterial
  • foot-and-mouth disease
  • antigen
  • virus
Date 2012-09-07
Availability unrestricted
Abstract
The suitability of a plant-based transient expression system using the agro-infiltration technique was compared to an Escherichia coli (E. coli)-based expression system to produce the VP1 protein from Serotype O, South Korean strain, of the foot-and mouth disease virus (FMDV). The full-length VP1 coding sequence was expressed in Escherichia coli as a fusion protein and purified as a His-tagged VP1 fusion protein with a yield of 14 mg L-1 bacterial culture. For transient expression in tobacco, the VP1 coding sequence was cloned into binary vector pMYV497, containing a CTB (cholera toxin B subunit) signal peptide and SEKDEL ER retention signal, and transiently agro-infiltrated into non-transgenic N. benthamiana and transgenic N. tabacum plants constitutively expressing the rice cysteine protease inhibitor OC-I. A protein resembling VP1 was detected using immuno-blotting analysis in both N. benthamiana and OC-I N. tabacum plants seven days post agro-infiltration. Although a possible stabilizing effect on VP1 was found due to OC-I expression, protein yields were not significantly different between transformed OC-I and non-OC-I control plants. Also, simultaneous co-infiltration with a plasmid allowing additional transient OC-I expression did not significantly improve VP1 production. The average VP1 amount achieved in OC-I expressing plants was 0.75% of total soluble protein. Overall, this study has shown that transient VP1 expression in tobacco is possible, but requiring further optimization, and that OC-I might have a stabilizing effect against proteolytic degradation of VP1 during advanced stages of senescence in agro-infiltrated plants coinciding with peaks in protein expression.

Copyright 2012 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:

Pillay, P 2012, Expression of the VP1 antigen from foot-and-mouth disease virus in a bacterial and plant-based expression system, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-08302012-103144 / >

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