Evaluation of DNA vaccines against Mycoplasma nasistruthionis sp. nov. str. Ms03 infections in ostriches and the production of IgA heavy chain proteins
| dc.contributor.advisor | Botes, Annelise | en_ZA |
| dc.contributor.author | Jonker, Hester Isabella | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Science. Dept. of Biochemistry. | en_ZA |
| dc.date.accessioned | 2017-02-21T10:28:12Z | |
| dc.date.accessioned | 2017-03-29T21:03:34Z | |
| dc.date.available | 2020-03-31T03:00:09Z | |
| dc.date.issued | 2017-03 | |
| dc.description | Thesis (MSc)--Stellenbosch University, 2017. | en_ZA |
| dc.description.abstract | ENGLISH ABSTRACT: Ostrich products have internationally become very popular with South Africa being the world leader in this industry. An increase in the demand for ostrich products has influenced production strategies by intensifying rearing conditions through the use of feedlot systems. Intensive rearing creates ideal conditions for the spread of pathogens such as mycoplasmas, which is associated with a respiratory disease syndrome amongst feedlot ostriches. The three ostrich-infecting mycoplasmas, Ms01, Ms02 and Ms03, together with other secondary pathogens result in reduced production. Since there are no vaccines available against these ostrich-infecting mycoplasmas, three DNA vaccines have been developed in this laboratory. Each vaccine consisted of a eukaryotic expression vector (pCI-neo, VR1020 or VR1012) containing the Ms03 oppA gene as antigen. The first objective of this study was to re-evaluate the anti-OppA immune response elicited by the pCI-neo and VR1020vaccines in ostriches. A vaccination trial was conducted and both vaccines were administered intramuscularly at 100, 600 and 1200 μg/ml doses followed by a booster vaccination. The anti-OppA immune response elicited by these vaccines in the ostriches was measured by means of the ELISA technique. All of the VR1020 vaccine doses as well as the 100 and 600 μg/ml doses of the pCI-neo vaccine were able to elicit a statistically significant anti-OppA immune response after administration of a booster vaccination. Since mycoplasmas target the respiratory system of ostriches a mucosally administered vaccine should also be considered. Opposed to the intramuscular route of vaccination, which results in humoral immunity represented predominantly by IgG, mucosal administration would result in mucosal immunity represented by IgA production. For the measurement of IgA production, the ELISA requires secondary anit-IgA antibodies. Although the whole antibody is typically used for the production of secondary antibodies, it is possible to use only the region representing the heavy chain constant region. This can then be produced as a recombinant protein that will allow an easy reproducible source for the production of secondary antibodies. The second objective of this study was therefore to evaluate the baculovirus-insect expression system for the production of the ostrich IgA heavy chain constant region (IgAH) protein, as this system will allow glycosylation of the protein product. The IgAH gene was inserted into the pAB-6xHis transfer plasmid and together with the ProFold-ER1 baculovirus used to co-transfect Sf9 insect cells for the production of ProFold-ER1_IgAH by means of homologous recombination. The IgAH protein was successfully expressed as confirmed by using HisProbe-HRP as well as previously produced rabbit anti-ostrich IgA antibodies during western blot analysis. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Volstruisprodukte het internasionaal baie gewild geword en Suid-Afrika is die wêreld leier in hierdie industrie. Die verhoogde aanvraag na volstruisprodukte het 'n invloed op produksie strategieë deur intesivisering van grootmaak kondisies deur die gebruik van voerkrale. Intensiewe grootmaak kondisies skep ideale toestande vir die verspreiding van patogene soos mikoplasmas wat geassosieer word met ‘n respiratoriese siektesindroom in voerkraalvolstruise. Drie volstruis-infekterende mikoplasmas, Ms01, Ms02 en Ms03, tesame met ander sekondêre patogene het 'n afname in produksie tot gevolg. Aangesien daar geen entstof teen hierdie volstruis-infekterende mikoplasmas beskikbaar is nie, is daar drie DNS entstowwe in hierdie laboratorium ontwikkel. Elke entsof bestaan uit ‘n eukariotiese ekspressievektor (pCI-neo, VR1020 of VR1012) wat die Ms03 oppA geen bevat wat dien as antigeen. Die eerste doelwit van hierdie studie was om die anti-OppA immuunreaksie, soos ontlok deur die pCI-neo en VR1020 entstowwe, te herevalueer. 'n Inentingsproef was deurgevoer waar beide entstowwe binnespiers toegedien was aan volstruise met onderskeidelik 'n 100, 600 en 1200 μg/ml dosis, gevolg deur 'n skraagdosis. Die gevolglike anti-OppA immuunreaksie was gemeet deur die ELISA tegniek. Alle VR1020 entstof dosisse asook die pCI-neo entstof se 100 en 600 μg/ml dosisse was daartoe in staat om 'n anti-OppA immuunreaksie te ontlok na die skraagdosis toediening. Aangesien mikoplasmas die respiratoriese sisteem van volstruise teiken, moet die ondersoek na 'n mukosale entstof oorweeg word. In vergelyking met binnespierse toediening van ‘n entstof wat sal lei tot humorale immuniteit wat oorwegend verteenwoordig word deur IgG, sal mukosale toediening lei tot mukosale immuniteit wat verteenwoordig word deur IgA produksie. Vir die meting van IgA produksie benodig die ELISA sekondêre anti-IgA teenliggaampies. Alhoewel die teenliggampie normaalweg in geheel gebruik word vir die produksie van sekondêre teenliggaampies, is dit moontlik om slegs die gedeelte wat die swaarketting konstante area verteenwoordig te gebruik. Dit kan dan as ‘n rekombinante proteïen geproduseer word wat sal dien as 'n maklike reproduseerbare bron vir die produksie van sekondêre teenliggaampies. Die tweede doelwit van hierdie studie was dus om die baculovirus-insek ekspressiesisteem te evalueer vir die produksie van die volstruis IgA swaarketting konstante area (IgAH) proteïen aangesien die sisteem glikosilering van die proteïen toelaat. Die IgAH geen was ingevoeg in die pAB-6xHis oordragplasmied en tesame met die ProFold-ER1 baculovirus gebruik om Sf9 insekselle te infekteer vir die produksie van ProFold-ER1_ IgAH deur middel van homoloë rekombinasie. Die IgAH proteïen was suksesvol uitgedruk soos bevestig deur HisProbe_HRP en voorheen geproduseerde konyn anti-volstruis IgA teenliggampies deur middel van 'n western-klad. | af_ZA |
| dc.description.version | Masters | en_ZA |
| dc.embargo.terms | 2020-03-31 | |
| dc.format.extent | x, 137 pages : illustrations (some color) | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/101461 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.subject | DNA vaccines | en_ZA |
| dc.subject | Ostriches -- Diseases -- South Africa | en_ZA |
| dc.subject | Antibody diversity | en_ZA |
| dc.subject | Ostrich products industry -- South Africa | en_ZA |
| dc.subject | Immunoglobulin A | en_ZA |
| dc.subject | Immunoglobulins -- Analyses | en_ZA |
| dc.title | Evaluation of DNA vaccines against Mycoplasma nasistruthionis sp. nov. str. Ms03 infections in ostriches and the production of IgA heavy chain proteins | en_ZA |
| dc.type | Thesis | en_ZA |