The indentification, contiguous sequence annotation, cloning and site-directed mutagenesis of the P100 vaccine candidate gene of the ostrich mycoplasma Ms02

[摘要] ENGLISH ABSTRACT:The ostrich industry in South Africa is currently threatened by respiratory disease in feedlot ostricheswhich causes a dramatic loss in production. Ms01, Ms02 and Ms03 were identified as the three ostrichspecificmycoplasmas to be associated with this respiratory disease in ostriches of South Africa. Theostrich-specific mycoplasmas have a major impact on ostrich production and for this reason there is aserious need for treatment for these infections.For this reason, the ostrich industry has undertaken an investigation into the development of vaccinesagainst mycoplasma infections. In this study, an approach to DNA vaccine development will beinvestigated and applied, specifically for the ostrich mycoplasma Ms02. Firstly, the whole genome ofMs02 was sequenced using GS FLX sequencing technology. The contiguous sequences obtained from thewhole-genome sequencing were analysed bioinformatically which included the annotation of thecontiguous sequences and the subsequent search for a vaccine candidate gene for the development of aDNA vaccine. The P100 gene of Ms02, which showed a high degree of homology with the P100 gene ofthe human pathogen M. hominis, was chosen as a vaccine candidate gene for the development of a DNAvaccine. The P100 gene was successfully cloned and subsequently modified by means of site-directedmutagenesis to correct for alternative codon usage, where after the modified P100 gene was subclonedinto the mammalian expression vector, pCI-neo for vaccination trials in the near future.

[发布机构] Stellenbosch University