Affiliation:
1. National Animal Disease Center, USDA Agricultural Research Service, Ames, Iowa 50010
2. Biomedical Genomics Center and Departments of Microbiology and Veterinary Pathobiology, University of Minnesota, Minneapolis, Minnesota 55488
Abstract
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.
Publisher
American Society for Microbiology
Cited by
44 articles.
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