Expression and immunogenicity of proteins encoded by sequences specific to Mycobacterium avium subsp. paratuberculosis

Abstract

The development of immunoassays specific for the diagnosis of Johne's disease in cattle requires antigens specific to Mycobacterium avium subsp. paratuberculosis. However, because of genetic similarity to other mycobacteria comprising the M. avium complex, no such antigens have been found. Through a comparative genomics approach, 21 potential coding sequences of M. avium subsp. paratuberculosis that 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 these M. avium subsp. paratuberculosis-specific sequences in Escherichia 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. avium subsp. paratuberculosis. These studies showed that 5 of the 21 gene products are produced by M. avium subsp. paratuberculosis and are antigenic. Immunoblot analysis with a panel of sera from 9 healthy cattle and 10 cattle with clinical disease shows that the same five M. avium subsp. paratuberculosis proteins are also detected within the context of infection. Collectively, these studies have used a genomic approach to identify novel M. avium subsp. paratuberculosis antigens that are not present in any other mycobacteria. These findings may have a major impact on improved diagnostics for Johne's disease.

FIG. 1.

SDS-PAGE and immunoblot analysis of affinity-purified M. avium subsp. paratuberculosis gene products. (A) Shown are two SDS-12% PAGE gels loaded with affinity-purified MBP fusion proteins that were stained with GelCode Blue. Size standards (in kilodaltons) are indicated on the left. (B through E) Corresponding immunoblots were probed with a monoclonal antibody to MBP (B) or with serum from rabbit 275 (C), mouse 47 (D), and clinical cow 67 (E). Note that MBP is not detected in panel C, D, or E. Lanes: 1, protein size standards; 2, gene 255; 3, gene 253; 4, gene 251; 5, gene 159; 6, gene 228; 7, gene 10; 8, gene 219; 9, gene 38; 10, gene 56; 11, gene 57; 12, MBP-MMP; 13, gene 218 (N-terminal half); 14, protein size standards; 15, gene 241; 16, gene 252; 17, gene 240; 18, gene 256; 19, gene 135; 20, gene 217; 21, gene 257; 22, gene 250; 23, gene 254; 24, gene 11; 25, gene 218 (C-terminal half); 26, MBP. Arrows point to the five subspecies-specific antigens identified in this study.

FIG. 2.

Expression of M. avium subsp. paratuberculosis six-His tag clones in E. coli. An immunoblot containing IPTG-induced E. coli BL21(DE3) lysates was probed with a monoclonal antibody that detects the polyhistidine tag. Size standards (in kilodaltons) are indicated on the left. Lanes: 1, protein size standards; 2, gene 254; 3, gene 159; 4, gene 56; 5, gene 38; 6, gene 228; 7, E. coli BL21(DE3) cells.