Expression and Immunogenicity of Proteins Encoded by Sequences Specific to<i>Mycobacterium avium</i>subsp.<i>paratuberculosis</i>

Bannantine, John P.; Hansen, Janis K.; Paustian, Michael L.; Amonsin, Alongkorn; Li, Fulin; Stabel, Judith R.; Kapur, Vivek

doi:10.1128/jcm.42.1.106-114.2004

Cited by 41 publications

(41 citation statements)

References 36 publications

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“…avium 104 genome (http://tigrblast.tigr .org/ufmg/). In addition, they are different from the 21 predicted coding sequences identified previously in our laboratory (1,2). PCR amplification using genomic DNA from 39 different isolates of M. avium subsp.…”

Section: Resultsmentioning

confidence: 99%

“…NC_002944), duplicated ORFs were eliminated and the number of unique ORFs was reduced to 34. Some of these ORFs have been previously characterized in our lab (1,2). Of the remaining ORFs, 13 were successfully amplified via PCR from M. avium subsp.…”

Section: Methodsmentioning

confidence: 99%

“…Similarly, BALB/c mice were immunized with a sonicated lysate of M. avium subsp. paratuberculosis K10 as previously described (1). Sera from six well-characterized cattle in the clinical stage of Johne's disease and three control cattle were used in immunoblot analysis for detection of antibodies that bind unique M. avium subsp.…”

Section: Methodsmentioning

confidence: 99%

“…paratuberculosis sequences have already been identified by our laboratory and others using a comparative genomic approach (2) and subtractive hybridization (14). Expression and analysis of these proteins has recently identified five candidate antigens (1). In this study, we have compared the recently completed, publicly available, but unpublished genome sequences of M. avium subsp.…”

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confidence: 99%

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Characterization of Novel Coding Sequences Specific toMycobacterium aviumsubsp.paratuberculosis: Implications for Diagnosis of Johne's Disease

et al. 2004

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Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's Disease, an economically important intestinal ailment of ruminants. Due to the considerable genetic and serologic cross-reactivity with closely related and ubiquitous members of the M. avium complex, a species-specific method for the serological diagnosis of Johne's disease is unavailable. Computational and PCR-based analysis of the complete genome sequence of M. avium subsp. paratuberculosis led to the identification of 13 open reading frames with no identifiable homologs. One of these sequences is a putative insertion element present in six copies on the M. avium subsp. paratuberculosis genome. These novel M. avium subsp. paratuberculosis genes were cloned into Escherichia coli expression vectors, and nine were successfully expressed as recombinant fusion proteins. Five of these proteins were purified in sufficient amounts to allow immunoblot analyses of their reactivity with sera from naturally infected cattle as well as mice and rabbits exposed to M. avium subsp. paratuberculosis. Fusion proteins representing MAP0862, MAP3732c, and MAP2963c were recognized by nearly all of the sera tested, including those from cattle in the clinical stages of disease. Notably, further analysis of the protein encoded by MAP0862 showed that it reacted with sera from additional infected cattle but not with sera from uninfected control animals. The fusion product of MAP0860c did not react with any of the sera tested, while the remaining four proteins were variably recognized by sera from M. avium subsp. paratuberculosis-infected cattle. Collectively, the results of this study demonstrate the utility of genomic data to identify potential diagnostic sequences.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of Novel Coding Sequences Specific toMycobacterium aviumsubsp.paratuberculosis: Implications for Diagnosis of Johne's Disease

et al. 2004

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“…The TAP expression system is essentially a screening tool and allows expression of proteins in relatively small quantities. In order to test these antigens by ELISA, the MAP1272c and MAP2121c proteins were expressed in E. coli, using the pMAL-c2 vector (New England Biolabs, Ipswich, MA) as previously described (2). Seven positive and two negative individual serum samples obtained from cattle that were confirmed to be positive or negative for Johne's disease by bacterial isolation were tested by ELISA.…”

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confidence: 99%

Rapid Expression of Mycobacterium avium subsp. paratuberculosis Recombinant Proteins for Antigen Discovery

Munir

Bannantine

et al. 2007

Clin Vaccine Immunol

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Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease, a chronic granulomatous enteritis of ruminants and other species. Detection of infection in animals is hampered by the lack of sensitive and specific diagnostic assays. We describe here an approach that utilizes translationally active PCR fragments for the rapid in vitro transcription and translation of recombinant proteins for antigen discovery in M. avium subsp. paratuberculosis. The investigations showed that the MAP1272c protein selectively reacts with sera from Johne's disease-positive cattle and represents an antigen of potential utility in M. avium subsp. paratuberculosis immunodiagnostics.Johne's disease is a chronic gastrointestinal inflammatory disease caused by Mycobacterium avium subsp. paratuberculosis (reviewed in reference 5). The disease occurs in wild and domestic ruminants, including dairy cattle, and has considerable impact on the global agricultural economy. The slow growth of the organism in laboratory culture and its extensive genetic relatedness with Mycobacterium avium subsp. avium (4, 7) have hindered diagnosis of Johne's disease using methods such as bacterial isolation, genomic assays, and serology. Recent investigations showed that the current enzyme-linked immunosorbent assay (ELISA)-based immunoassays have poor sensitivity, detecting fewer than one-third of all infected cattle (6). Furthermore, the use of crude M. avium subsp. paratuberculosis protein mixtures as antigens compromises assay specificity due to conservation of proteins across Mycobacterium avium complex organisms. Hence, identification of suitable M. avium subsp. paratuberculosis antigens that could enable early, sensitive, and specific detection of M. avium subsp. paratuberculosis infection is critically needed to facilitate adequate disease control measures.In order to capitalize on the availability of the complete genome sequence of M. avium subsp. paratuberculosis (10) for novel antigen discovery, we describe here the application of an in vitro transcription and translation system that enables expression of M. avium subsp. paratuberculosis recombinant proteins directly from transcriptionally active PCR (TAP) fragments (11). This approach obviates the need for cloning of individual genes and expression of proteins in a heterologous system. It is also amenable for adaptation to a high-throughput format and enables the expression of hundreds of genes in days versus the months needed for cloning-based expression. Hence, this method is labor-, time-, and cost-effective and is ideal for large-scale antigen discovery.In order to evaluate the utility of this approach for rapid expression and screening of potential antigens for use in M. avium subsp. paratuberculosis immunodiagnostics, we chose two candidate M. avium subsp. paratuberculosis open reading frames (ORFs), MAP1272c and MAP2121c, that have not previously been characterized either functionally or immunologically. Preliminary computational and comparative genomic analyses suggest...

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Identification of proteins of potential diagnostic value for bovine paratuberculosis

2006

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Previously we showed that Mycobacterium paratuberculosis culture filtrates (CFs) contain more antigens that react with sera from infected cattle than do cellular extracts of the organism. The goal of the present study was to identify proteins of potential diagnostic value among these CF proteins. Proteins of potential interest were first separated by 2-DE. Roughly 240 CF protein spots were detected on CBB-stained gels using Phoretix 2D software. Of these, 83% reacted with serum from M. paratuberculosis-infected cattle in immunoblots. When bovine serum was absorbed with M. phlei antigens, however, only 37 of these antigenic protein spots were reactive. Twenty-four of these spots were selected for identification based on their immunoblot staining intensity and differences in pI and mass. A total of 14 proteins were ultimately identified by MS and BLAST searches as ModD, PepA, ArgJ, CobT, Antigen 85C, and nine hypothetical proteins. N-terminal peptide analysis of PepA, Antigen 85C, ModD, MAP1693c, MAP2168c, and MAP1022c showed that each protein has 27-39 amino acids that may function as a signal sequence suggesting they are secreted through a Sec-dependent pathway. These 14 proteins from M. paratuberculosis CF are strong candidates for use as antigens for improved serodiagnostic tests for bovine paratuberculosis.

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

Cited by 41 publications

References 36 publications

Characterization of Novel Coding Sequences Specific toMycobacterium aviumsubsp.paratuberculosis: Implications for Diagnosis of Johne's Disease

Characterization of Novel Coding Sequences Specific toMycobacterium aviumsubsp.paratuberculosis: Implications for Diagnosis of Johne's Disease

Rapid Expression of Mycobacterium avium subsp. paratuberculosis Recombinant Proteins for Antigen Discovery

Identification of proteins of potential diagnostic value for bovine paratuberculosis

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