|Alongkorn, Amonsin - UNIV OF MN|
|Kapur, Vivek - UNIV OF MN|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: September 28, 2003
Publication Date: N/A
Technical Abstract: The development of immunoassays specific for the diagnosis of Johne's disease in cattle requires antigens specific to Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis). However, because of genetic similarity to other mycobacteria comprising the MAC complex, no such antigens have been found. Through a comparative genomics approach, we have previously identified 21 M. paratuberculosis potential coding sequences that are not represented in any other mycobacterial species tested (Bannantine et al. J. Clin. Microbiol. 40:1303-1310). With the recent completion of the genome sequence, an additional 14 M. paratuberculosis-specific sequences have subsequently been found. We describe the cloning, heterologous expression, and antigenic analysis of these M. paratuberculosis-specific sequences in E. coli. Nucleotide sequences representing each unique predicted coding region were amplified and cloned into two different E. coli expression vectors encoding polyhistidine or maltose binding protein (MBP) affinity-purification tags. Purified fusion proteins were isolated under denaturing conditions and were evaluated in immunoblotting studies with sera from rabbits and mice immunized with M. paratuberculosis. These studies showed that eight of the 35 gene products are produced by M. paratuberculosis and are antigenic. Immunoblot analysis with a panel of sera from nine healthy and ten clinical cattle shows the same eight M. paratuberculosis proteins are also detected within the context of infection. Collectively, these studies have used a genomic approach to identify novel M. paratuberculosis antigens that are not present in any other mycobacteria. These findings may have a major impact on improved diagnostics for Johne's disease.