Identification and characterization of an 8.4 kDa protein antigen of Mycobacterium bovis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand

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Massey University
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The culture filtrate (CF) derived from a M. smegmatis subclone transformed with the mycobacteria/E. coli plasmid shuttle vector pSU4511 containing a 4.3 kb fragment of M. bovis DNA (M. smegmatis pSU151.43), was observed to stimulate PBMC from a steer vaccinated with M. bovis BCG to proliferate and produce IFN-γ. To identify the source of immunoreactivity, the proteins in CF derived from M. smegmatis pSU151.43 were separated by fast protein liquid chromotography (FPLC) and the fractions were screened in whole blood IFN-γ assays. A stimulatory protein was purified that had a molecular mass of 8335 Da and the N-terminal amino acid sequence: DPVDAVINTT. Polyclonal antisera were raised against the purified recombinant antigen in rabbits and used for Western blotting. The nucleotide sequence of the 4.3 kb insert of M. bovis DNA was determined, and the open reading frame (ORF) coding for the 8.4 kDa protein was identified. Computer analysis of the deduced amino acid sequence with the programme PSORT predicted that the nascent protein consisted of a 28 amino acid export signal sequence followed by an 82 amino acid mature protein. It was also found that M. avium possesses a nucleotide sequence that potentially codes for a protein with a high degree of homology to the 8.4 kDa antigen of M. bovis. A segment of the 4.3 kb insert of M. bovis DNA adjacent to the gene coding for the 8.4 kDa antigen was found to be polymorphic between the strain of M. bovis from which the cosmid library was constructed and the published sequence of M. tuberculosis H37Rv (Cole et al. 1998). The M. bovis sequence contained 1.7 copies of a 62 bp exact tandem repeat and the M. tuberculosis sequence contained 2.7 copies. The species distribution of the 62 bp exact tandem repeat (ETR) locus was characterized by polymerase chain reaction (PCR) and Southern blotting. The 62 bp ETR was found to occur only in M. tuberculosis complex species and may be a useful genetic marker for differentiating between M. bovis and M. tuberculosis. Lymphocyte proliferation and IFN-γ assays were used to measure the responses of ten BCG vaccinated and ten unvaccinated calves the 8.4 kDa antigen, PPD-B and PPD-A tuberculins, both before and after intratracheal challenge infection with virulent M. bovis. The results provided evidence that vaccination of cattle with M. bovis BCG but not infection with M. bovis appeared to elicit an immune response to the 8.4 kDa antigen of M. bovis. To obtain greater quantities of recombinant 8.4 kDa antigen, the gene that codes for the protein was cloned into E. coli and M. smegmatis expression plasmids. The 8.4 kDa antigen was overexpressed and secreted with an N-terminal 6 x Histidine tag by M. smegmatis. Approximately 500 µg of 6 x Histidine tagged 8.4 kDa Ag were purified / litre of CF in one step by metal chelate affinity chromatography. The recombinant protein was shown to elicit specific IFN-γ responses in vitro.
Antigen analysis, Gene expression