Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Presently, the observed variation in symptoms of Potato leafroll virus (PLRV) infection in potato
cultivars in South Africa cannot be reconciled with PLRV symptoms obtained 10-15 years ago,
even if the different interactions between the pathogen and the cultivar are taken into account.
In an effort to analyze this variation, mutations in the coat protein (CP) gene of South African
isolates of PLRV were assessed. The CP gene of PLRV isolates from different areas within
South Africa was amplified by reverse transcription-polymerase chain reaction (RT-PCR),
cloned and sequenced. Significant sequence variation in the CP gene was found within the
analyzed South African isolates of PLRV. Phylogenetic analysis revealed two major clades with
most South African isolates and an Australian and North American isolate grouped together and
the remainder grouped with isolates from diverse countries worldwide. The deduced amino acid
sequences from representatives of these two clades indicated differences in CP threedimensional
structure.
In an effort to produce recombinant PLRV CP for the production of antibodies specific for South
African isolates of PLRV for use in enzyme-linked immunosorbent assay (ELISA), the CP gene
of a South African isolate of PLRV was subcloned into a bacterial expression vector (pET14-b).
Expression of full length recombinant PLRV CP was attempted in Escherichia coli strains
BL21(DE3)pLysS, Rosetta-gami B(DE3)pLysS and Rosetta-2(DE3)pLysS. As this was not
successful, the PLRV CP gene was subcloned in another expression vector (pGEX) for
expression as an N-terminal fusion protein with glutathione-S-transferase (GST) in E. coli
strains BL21(DE3)pLysS and Rosetta-2(DE3)pLysS. The recombinant GST-PLRV CP fusion
protein was purified and used for antibody production in rabbits. Using western blots, the
effectiveness of antibodies produced to recombinant GST-PLRV CP fusion protein was
assessed for PLRV recognition. It was found that antibodies to the recombinant GST-PLRV CP
fusion protein were more effective for the detection of GST than PLRV CP and that production
of antibodies to the cleaved PLRV CP product would be necessary if antibodies are required for
ELISA applications. / AFRIKAANSE OPSOMMING: Huidiglik kan die waargeneemde simptome van infeksie met aartappelrolbladvirus (Potato
leafroll virus, PLRV) in aartappelkultivars in Suid-Afrika nie vereenselwig word met PLRV
simptome wat 10-15 jaar gelede verkry was nie, selfs al word die verskillende interaksies tussen
die patogeen en kultivar in ag geneem.
In ‘n poging om hierdie variasie te analiseer, was mutasies in die mantelproteïen (CP) geen van
Suid-Afrikaanse isolate van PLRV bepaal. Die CP geen van PLRV isolate van verskillende
areas in Suid-Afrika was ge-amplifiseer met behulp van die tru transkripsie-polimerase ketting
reaksie (RT-PCR), gekloneer en die nukleotiedvolgorde bepaal. Noemenswaardige nukleotied
variasie is in die CP gene van die ge-analiseerde Suid-Afrikaanse isolate van PLRV gevind.
Filogenetiese analises het gedui op twee hoof klades met die meeste van die Suid-Afrikaanse
isolate wat saam met ‘n Australiese en Noord-Amerikaanse isolaat gegroepeer en die res wat
met isolate van verskillende lande wêreldwyd gegroepeer. Die afgeleide aminosuurvolgordes
van verteenwoordigers van bogenoemde twee klades het gedui op verskille in die CP driedimensionele
struktuur.
In ‘n poging om rekombinante PLRV CP te produseer vir die produksie van antiliggame
spesifiek teen Suid-Afrikaanse isolate van PLRV om in “enzyme-linked immunosorbent assay”
(ELISA) te gebruik, was die CP geen van ‘n Suid-Afrikaanse isolaat van PLRV gesubkloneer in
‘n bakteriële ekspressie vektor (pET14-b). Daar was gepoog om vollengte rekombinante PLRV
CP in die Escherichia coli rasse BL21(DE3)pLysS, Rosetta-gami B(DE3)pLysS en Rosetta-
2(DE3)pLysS te produseer. Aangesien dit nie suksesvol was nie, was die PLRV CP
gesubkloneer in ‘n ander ekspressie vektor (pGEX) sodat die proteïen as ‘n N-terminale fusie
proteïen met “glutathione-S-transferase” (GST) in E. coli rasse BL21(DE3)pLysS en Rosetta-
2(DE3)pLysS geproduseer kon word. Die rekombinante GST-PLRV CP fusie proteïen was
gesuiwer en gebruik vir antiliggaam produksie in konyne. Die effektiwiteit van die antiliggame
wat teen rekombinante GST-PLRV CP fusie proteïen geproduseer was vir PLRV herkenning is
deur middel van “western blots” geanaliseer. Dit was gevind dat antiliggame teen die
rekombinante GST-PLRV CP fusie proteïen meer effektief was vir die herkenning van GST as
PLRV CP. Gevolglik sal dit nodig wees om antiliggame teen die gesnyde PLRV CP produk te
maak vir gebruik in ELISA.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/19861 |
| Date | 03 1900 |
| Creators | Rothmann, Adri Hilda |
| Contributors | Bellstedt, D.U., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 124 leaves : ill. (some col.) |
| Rights | Stellenbosch University |
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