Submitted to: Journal of Clinical Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/26/2003
Publication Date: 1/1/2004
Citation: Bannantine, J.P., Hansen, J.K., Paustian, M., Amonsin, A., Li, L.L., Stabel, J.R., Kapur, V. 2004. Expressionand immunogenicity of proteins encoded by sequences specific to mycobacterium avium subsp. paratuberculosis. Journal of Clinical Microbiology. 42:106-114. Interpretive Summary: The goal of this study was to find proteins that are produced only by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), because these proteins may be used in a specific diagnostic test for Johne's disease. By comparing the available genome sequences of mycobacterial species, we were able to identify 21 unique proteins. All 21 proteins were produced and purified from recombinant E. coli cells. These studies showed that five of the 21 proteins are detected by sera from animals with Johne's disease. These findings may have a major impact on improved diagnostics for Johne's disease.
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 (n=9; Bannantine et al. J. Clin. Microbiol. 40:1303-1310). Here 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. All 21 of the MBP fusion proteins were successfully purified under denaturing conditions and were evaluated in immunoblotting studies with sera from rabbits and mice immunized with M. paratuberculosis. These studies showed that five of the 21 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 five 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.