Document Type Master's Dissertation Author Pillay, Priyen firstname.lastname@example.org 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
- foot-and-mouth disease
Date 2012-09-07 Availability unrestricted AbstractThe 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.
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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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