Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The South African ostrich industry is under enormous threats due to diseases contracted by the ostriches. H5N2
virus (avian influenza) outbreaks the past two years have resulted in thousands of ostriches having to be culled.
However, the more silent respiratory infectious agents of ostriches are the three ostrich-specific mycoplasmas.
Named Ms01, Ms02, and Ms03, these three mycoplasmas are responsible for dramatic production losses each
year, due to their intrusive nature and the fact that no vaccines are currently available to prevent mycoplasma
infections in ostriches. The use of antibiotics does not eradicate the disease completely, but only alleviates
symptoms. The ostrich industry commissioned investigations into the development of three specific vaccines
using the relatively novel approach of DNA vaccination.
The concept of DNA vaccine development is based on the availability of complete genome sequences of the
pathogen against which the vaccine is to be developed. This is necessary in order to identify vaccine candidate
genes through comparative genomic studies. The Ms02 genome has previously been sequenced, resulting in
28 large contiguous sequences. This thesis used the technique of Thermal Asymmetric Interlaced Polymerase
Chain Reaction (TAIL-PCR) to attempt assembly of these 28 contiguous sequences. The number was reduced
to 14 large contiguous sequences, which were then subjected to repetitive sequence analysis and open reading
frame analysis. Bioinformatic software was also used to predict the origin of replication. The extent of repeats in
the Ms02 genome is illustrated, as well as the problems with genome assembly when dealing with repetitive-rich
and A+T-rich genomes as those of mycoplasmas.
Previous studies determined the mycoplasma oppA gene to be a good vaccine candidate gene, due to its
cytadherent properties. This thesis describes the development of three DNA vaccines containing the Ms02
oppA gene, and a preliminary attempt to prove expression of one of these vaccines in a cell culture-based
system. The DNA vaccine vectors pCI-neo, VR1012, and VR1020 were chosen for the vaccine development.
The Ms02 oppA gene was also cloned into the prokaryotic expression vector pGEX-4T-1 in order to express the
OppA protein for purification. The purified protein may be used in future serological tests in ostrich vaccination
trials. In this study the protein was used to elicit anti-OppA rabbit antibodies, which were used to attempt
detection of the pCI-neo-driven OppA protein expression in an MDA cell line in a transfection study. However,
preliminary findings could not detect expression, but did indicate that the currently used colorimetric western blot
technique may not be sensitive enough. It is suggested that different cell lines need to be investigated. Further
optimisations are also required to decrease the observed non-specific binding. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse volstruisbedryf is onder geweldige druk vanweë siektes wat die volstruise bedreig. Die
epidemie van die H5N2 virus (voëlgriep) in die afgelope twee jaar het veroorsaak dat duisende volstruise van
kant gemaak moes word. Daar is egter nog ‘n bedreiging wat tot geweldige produksie verliese lei elke jaar: die
respiratoriese infeksies wat versoorsaak word deur die drie volstruis mikoplasmas, genoem Ms01, Ms02 en
Ms03. Geen entstowwe is tans beskibaar om die infeksies te voorkom nie, en behandeling met behulp van
antibiotikas is nie effektief in die genesing van infeksie nie, maar help net om die simptome te verlig. Weens die
erns van die saak, het die Suid-Afrikaanse volstruisbedryf ‘n ondersoek geloods na die ontwikkeling van
enstowwe teen elkeen van die drie volstruis mikoplasmas. Die relatiewe nuwe benadering van DNA-entstof
ontwikkeling was die strategiese keuse.
Die beginsel van DNA-entstof ontwikkeling berus op die beskikbaarheid van die genoomvolgordes van die
siekte-veroorsakende organisme waarteen die enstof ontwikkel word. Geskikte kandidaat entstof gene word so
opgespoor met behulp van vergelykende studies met ander beskikbare genome. Die Ms02 genoomvolgorde is
voorheen bepaal en word verteenwoordig deur 28 groot geenvolgorde fragmente. Die tegniek van Thermal
Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR) is gebruik om van die 28 fragmente aan mekaar
te las. Die aantal fragmente is verminder na 14 groot geenvolgorde fragmente, wat vervolgens gebruik was om
die omvang van herhalende volgordes in die genoom te bepaal, om nuwe leesrame te ondersoek, asook om die
oorsprong van DNA replikasie op te spoor met behulp van bioinformatika sagteware. Die omvang van die
herhalende aard van die Ms02 genoom word geïllustreer, asook die gepaardgaande probleme met die las van
geenvolgorde fragmente wanneer met genome van veelvuldige herhalende volgordes, wat boonop A+T-ryk is,
gewerk word, soos die van mikoplasmas.
Vorige studies het die mikoplasma oppA geen geïdentifiseer as ‘n geskikte kandidaat entstof geen as gevolg van
sy selaanhegting-eienskappe. Hierdie studie behels die invoeging van die Ms02 oppA geen in drie DNA-enstof
vektore, naamlik pCI-neo, VR1012, en VR1020, asook die voorlopige poging om bewys van uitdrukking van een
van die entstowwe in ‘n selkultuursisteem te bewerkstellig. Die geen is ook gekloneer in die prokariotiese
ekspressie vektor pGEX-4T-1, ten einde die Ms02 OppA proteïen te isoleer. Die geïsoleerde proteïen kan in
serologiese toetse in toekomstige volstruis enstof proewe gebruik word. In hierdie studie is die proteïen gebruik
om konyn teenliggame teen dit op te wek, wat dan gebruik was om vir die pCI-neo-gedrewe ekspressie van die
oppA geen te toets in ‘n selkultuur omgewing deur ‘n MDA sellyn te transfekteer. Die voorlopige resultate toon
nie ekspressie van die OppA proteïen aan nie, maar het wel uitgelig dat die western blot tegniek wat tans
gebruik word, dalk nie sensitief genoeg is nie. Dit kan belowend wees om ander tipes selle te toets. Verdere
optimisering is ook nodig om die nie-spesifieke binding wat waargeneem is, te verlaag. / South African Ostrich Business Chamber
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/80381 |
| Date | 03 1900 |
| Creators | Strydom, Marliz |
| Contributors | Botes, A., 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 | Unknown |
| Type | Thesis |
| Format | ix, 141 p. : ill. (some col.) |
| Rights | Stellenbosch University |
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