| Popis: |
The work presented in this thesis was undertaken as part of a larger research initiative in the field of ruminant paratuberculosis (Johne's disease). One of the group's main research objectives was the identification and characterisation of subspecies-specific genes and proteins of the two members of the Mycobacterium avium complex (MAC) known to have a causal role in ruminant paratuberculosis, namely Mycobacterium avium spp. paratuberculosis (M. a. paratuberculosis) and IS901/902-positive strains of Mycobacterium avium (M. avium). A protein antigen of 40kDa (p40) was identified in an IS901/902-positive strain of M. avium, but could not be detected in M. a. paratuberculosis, IS901/902-negative M avium, or in any of 13 other species of Mycobacterium tested. Examination of 19 further MAC field isolates confirmed the absolute association between p40 and IS901/902, suggesting that p40 is a novel, subspecies-specific protein which may potentially be of value as a diagnostic antigen. Soluble p40 antigen was precipitated from cleared cell lysates and purified to homogeneity using a series of chromatographic separations. Chemical cleavage and Edman degradation of the purified p40 antigen provided both amino-terminal and internal amino acid sequence data which showed no sequence identity with any protein sequence currently in the OWL database. Translations of IS901/902 in all six reading frames revealed amino acid sequences which confirmed that the genomic insertion sequence does not encode the p40 antigen. A second protein of 30kDa (p30) was purified simultaneously, and analysis of the first 10 amino-terminal amino acid residues revealed up to 90% sequence identity with the mature secretory antigens of the "antigen 85 complex" of 8 other species of Mycobacterium. Experiments were designed to ascertain whether the p40 antigen was expressed in vivo and to assess whether any immune response to the p40 could be exploited in the development of an immune-based assay to differentiate between animals infected with M. a. paratuberculosis, and others infected with IS901/902-positive status of M. avium. |
