Rotavirus infection causes severe gastroenteritis, affecting all children under the age of five
regardless of hygiene or water quality. The currently licensed vaccines succeeded in
reducing diarrhoea worldwide, but they still have shortcomings, especially the efficacy of the
vaccines in developing countries. One of the main reasons for this can be due to the
difference in strains, since the strains used to develop the currently licensed vaccines
(RotaTeq and Rotarix) were selected from strains circulating in the developed world (G1, G2,
G3 and G4), while the main strains present in Africa (G8, G9 and G12) were not included. A
second shortcoming of the currently licensed vaccines is the cost of these vaccines. The
vaccines are very expensive and most developing countries cannot afford the vaccines as
well as the fact that the manufacturing companies cannot produce enough vaccines for all
the countries. An attractive alternative to the currently licensed rotavirus vaccines is the
non-live vaccine candidate, virus-like particles, which can provide a possible cheaper, safer
and efficacious alternative or complement the currently licensed vaccines.
Therefore, in this study a South African G9P[6] rotavirus strain, RVA/Humanwt/
ZAF/GR10924/1999/G9P[6], was used to determine whether or not co-expression of the
structural proteins VP2 (genome segment 2) and VP6 (genome segment 6) was possible in
bacteria and yeast. The South African GR10924 G9P[6] neonatal strain was previously
obtained from a stool sample and the nucleotide consensus sequence was determined for
both genome segment 2 (VP2) and genome segment 6 (VP6). Bacterial codon optimised
coding regions or open reading frames were used in this study. The open reading frames
(ORFs) of the genome segments encoding, VP2 and VP6, were cloned into the expression
vector pETDuet-1, which allows for the simultaneous expression of two genes in bacteria.
The ORF of genome segment 6 was purchased from GeneScript and the ORF of genome
segment 2 was obtained from Dr AC Potgieter (Deltamune (Pty) Ltd R&D, South Africa).
Compatible restriction enzyme sites were used to sub-clone the ORF of the bacterial codon
optimised genome segments into the expression vector. Only the expression of the VP6
protein in bacteria was observed with Coomassie stained SDS-PAGE.
The ORFs encoding VP2 (genome segment 2) and VP6 (genome segment 6) of the wild
type GR10924 G9P[6] strain were cloned into the wide range yeast expression system
vector, pKM173, which allows for the simultaneous expression of more than one gene.
Several yeast strains were used in this study namely Kluyveromyces marxianus,
Kluyveromyces lactis, Candida deformans, Saccharomyces cerevisiae, Yarrowia lipolytica,
Arxula adeninivorans, Hansenula polymorpha and Debaryomyces hansenii. Expression of
both proteins was not detected in the several yeast strains, as seen with western blot analysis. DNA extractions were done on two colonies of each yeast strain that were used for
western blot analysis to evaluate successful integration into the yeast genomes. Only a few
of the colonies contained either both of the genome segments or only one of the two
genome segments of interest.
To summarise, the simultaneous expression of VP2 and VP6 from rotavirus GR10924
G9P[6] was not successful in bacteria or yeast, but it was possible to soluble express the
bacterial codon optimised GR10924 G9P[6] VP6 in bacteria using the pETDuet-1 as
expression vector. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015
| Identifer | oai:union.ndltd.org:NWUBOLOKA1/oai:dspace.nwu.ac.za:10394/16082 |
| Date | January 2015 |
| Creators | Naudé, Louisa Aletta |
| Source Sets | North-West University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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