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dc.contributor.advisorVan Dijk, A.A.
dc.contributor.advisorO'Neill, H.G.
dc.contributor.authorVan der Westhuizen, Maria Jacoba
dc.date.accessioned2014-01-07T14:05:38Z
dc.date.available2014-01-07T14:05:38Z
dc.date.issued2012
dc.identifier.urihttp://hdl.handle.net/10394/9852
dc.descriptionThesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.
dc.description.abstractRotavirus 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.en_US
dc.language.isoenen_US
dc.publisherNorth-West University
dc.subjectRotavirusen_US
dc.subjectgastroenteritisen_US
dc.subjectsub-Saharan Africaen_US
dc.subjectG9P[6]en_US
dc.subjectRotavirus-like particlesen_US
dc.subjecttransmission electron microscopyen_US
dc.subjectdouble-layered particlesen_US
dc.subjecttriple-layered particles,en_US
dc.subjectgastro-enteritisen_US
dc.subjectsub-Sahara Afrikaen_US
dc.subjectRotavirus virusagtige partikelsen_US
dc.subjectnie-lewendige entstofen_US
dc.subjectNonlive vaccineen_US
dc.subjectBaculovirus Expression Systemen_US
dc.subjectSpodoptera frugiperdaen_US
dc.subjectBaculovirus Uitdrukkings Stelselen_US
dc.subjecttransmissie elektron mikroskopieen_US
dc.subjectdubbellaag partikelsen_US
dc.subjecttrippellaag partikelsen_US
dc.titleThe engineering and optimization of expression of rotavirus–like particles in insect cells using a South African G9P[6] rotavirus strainen
dc.typeThesisen_US
dc.description.thesistypeMastersen_US
dc.contributor.researchID10997938 - Van Dijk, Alberdina Aike (Supervisor)
dc.contributor.researchID21484317 - O'Niel, Hester Gertruida (Supervisor)


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