Expression and Immunogenicity of Proteins Encoded by Sequences Specific toMycobacterium aviumsubsp.paratuberculosis

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Abstract
The development of immunoassays specific for the diagnosis of Johne's disease in cattle requires antigens specific toMycobacterium aviumsubsp.paratuberculosis. However, because of genetic similarity to other mycobacteria comprising theM. aviumcomplex, no such antigens have been found. Through a comparative genomics approach, 21 potential coding sequences ofM. aviumsubsp.paratuberculosisthat are not represented in any other mycobacterial species tested (n= 9) were previously identified (J. P. Bannantine, E. Baechler, Q. Zhang, L. Li, and V. Kapur, J. Clin. Microbiol.40:1303-1310, 2002). Here we describe the cloning, heterologous expression, and antigenic analysis of theseM. aviumsubsp.paratuberculosis-specific sequences inEscherichia coli. Nucleotide sequences representing each unique predicted coding region were amplified and cloned into two differentE. coliexpression 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 withM. aviumsubsp.paratuberculosis. These studies showed that 5 of the 21 gene products are produced byM. aviumsubsp.paratuberculosisand are antigenic. Immunoblot analysis with a panel of sera from 9 healthy cattle and 10 cattle with clinical disease shows that the same fiveM. aviumsubsp.paratuberculosisproteins are also detected within the context of infection. Collectively, these studies have used a genomic approach to identify novelM. aviumsubsp.paratuberculosisantigens that are not present in any other mycobacteria. These findings may have a major impact on improved diagnostics for Johne's disease.

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