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Cloning and evaluation of expression of the open reading frames of a South African G9P[6] rotavirus strain encoding rotavirus structural proteins VP2 and VP6 in bacteria and yeast / Louisa Aletta NaudéNaudé, Louisa Aletta January 2015 (has links)
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
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Cloning and evaluation of expression of the open reading frames of a South African G9P[6] rotavirus strain encoding rotavirus structural proteins VP2 and VP6 in bacteria and yeast / Louisa Aletta NaudéNaudé, Louisa Aletta January 2015 (has links)
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
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The engineering and optimization of expression of rotavirus-like particles in insect cells using a South African G9P[6] rotavirus strain / by Maria J. van der Westhuizen.Van der Westhuizen, Maria Jacoba January 2012 (has links)
Rotavirus infection causes gastroenteritis, specifically severe gastroenteritis, affecting children younger than five globally, regardless of hygiene and water quality. Current licensed, live, attenuated vaccines do not contain the G9 genotype, which is a prevalent rotavirus strain circulating in sub-Saharan Africa, a region that carries a high rotavirus disease burden. Rotavirus-like particles (RV-VLPs) is an attractive non-live vaccine candidate, which has shown promising results in animal studies. Previously, dsRNA was extracted from a stool sample containing a South African human G9P[6] neonatal strain, and amplified cDNA using a sequence-independent procedure. The consensus sequence was obtained for the genome segments using 454® pyrosequencing. The insect-cell-codon-optimized genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBACquad vector (pFBq). Several combinations of the genome segments were cloned to produce double-layered particles (DLP; pFBqVP2VP6) or triple-layered particles (TLP; pFBqVP2VP6VP7). In the current study, a ΔTLP (pFBqdVP2-VP8*VP6VP7) construct was generated. The first 92 amino acids of VP2 are not necessary for the conformation of recombinant RV-VLPs. The ORF of VP8*, which contains immune important epitopes, was fused to the 5’ end of the dVP2 coding region resulting in a dVP2-VP8* fused protein which was expressed in the presence of VP6 and VP7 to produce ΔTLPs. The Bac-to-Bac® Baculovirus Expression System and Spodoptera frugiperda (Sf) 9 insect cells were used for expression. All the proteins were successfully expressed. VP2, VP6, VP4 and the dVP2-VP8* fused protein were visible on Coomassie stained SDS-PAGE. Expression of VP7 could only be confirmed with western blot analysis. Particle formation, as assessed by transmission electron microscopy (TEM), was observed for DLPs. No TLPs of dVP2-8*/6/7 or VP2/6/7 were visualized due to the lower expression level of VP7 and the lack of calcium supplements during the assembly process. In conclusion, it was possible to produce RV-DLPs derived from the consensus sequence determined for a G9P[6] rotavirus directly from stool without prior propagation in cell culture or virus isolation. This strain contains both the G9 and P[6] genotypes that are currently prevalent in sub-Saharan Africa. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.
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The engineering and optimization of expression of rotavirus-like particles in insect cells using a South African G9P[6] rotavirus strain / by Maria J. van der Westhuizen.Van der Westhuizen, Maria Jacoba January 2012 (has links)
Rotavirus infection causes gastroenteritis, specifically severe gastroenteritis, affecting children younger than five globally, regardless of hygiene and water quality. Current licensed, live, attenuated vaccines do not contain the G9 genotype, which is a prevalent rotavirus strain circulating in sub-Saharan Africa, a region that carries a high rotavirus disease burden. Rotavirus-like particles (RV-VLPs) is an attractive non-live vaccine candidate, which has shown promising results in animal studies. Previously, dsRNA was extracted from a stool sample containing a South African human G9P[6] neonatal strain, and amplified cDNA using a sequence-independent procedure. The consensus sequence was obtained for the genome segments using 454® pyrosequencing. The insect-cell-codon-optimized genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBACquad vector (pFBq). Several combinations of the genome segments were cloned to produce double-layered particles (DLP; pFBqVP2VP6) or triple-layered particles (TLP; pFBqVP2VP6VP7). In the current study, a ΔTLP (pFBqdVP2-VP8*VP6VP7) construct was generated. The first 92 amino acids of VP2 are not necessary for the conformation of recombinant RV-VLPs. The ORF of VP8*, which contains immune important epitopes, was fused to the 5’ end of the dVP2 coding region resulting in a dVP2-VP8* fused protein which was expressed in the presence of VP6 and VP7 to produce ΔTLPs. The Bac-to-Bac® Baculovirus Expression System and Spodoptera frugiperda (Sf) 9 insect cells were used for expression. All the proteins were successfully expressed. VP2, VP6, VP4 and the dVP2-VP8* fused protein were visible on Coomassie stained SDS-PAGE. Expression of VP7 could only be confirmed with western blot analysis. Particle formation, as assessed by transmission electron microscopy (TEM), was observed for DLPs. No TLPs of dVP2-8*/6/7 or VP2/6/7 were visualized due to the lower expression level of VP7 and the lack of calcium supplements during the assembly process. In conclusion, it was possible to produce RV-DLPs derived from the consensus sequence determined for a G9P[6] rotavirus directly from stool without prior propagation in cell culture or virus isolation. This strain contains both the G9 and P[6] genotypes that are currently prevalent in sub-Saharan Africa. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.
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