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Cloning, expression, purification and serodiagnostic evaluation of fourteen Mycobacterium paratuberculosis proteins
Donghee Cho1, Sung-Jae Shin, Adel M Talaat
1School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA.
Abstract:
Fourteen proteins of potential diagnostic value for bovine paratuberculosis were identified in the culture filtrate of Mycobacterium paratuberculosis JTC303 by immunoblot and mass spectrometry. The goals of the present study were to express these 14 ORFs in Escherichia coli and evaluate their antigenicity. All 14 proteins were expressed in E. coli BL21(DE3) after transformation with the pET-22b(+) vector. Yields of insoluble proteins were higher than those of the soluble proteins. Polyclonal rabbit antibodies directed against culture filtrate of JTC303 strain confirmed that five of the expressed and purified proteins are culture filtrate components: ModD, Antigen 85C, PepA, MAP1693c, and MAP2168c. Evaluation of ModD as an ELISA solid-phase antigen on a set of bovine sera from well-characterized paratuberculosis cases and infection-free controls revealed that there was strong serum antibody reactivity to rModD in many infected cattle. However, the overall rModD ELISA sensitivity and specificity for bovine paratuberculosis was not greater than those of ELISAs using crude antigens such as cellular extract or culture filtrate for plate coating, as judged by area under the curve (AUC) of Receiver-operating curve (ROC) analysis. However, an ELISA using natural ModD as the solid-phase antigen had a higher sensitivity and AUC than did rModD suggesting diminution of antigenicity in rModD. Taken together, our results showed that the natural forms of the identified proteins may be useful for diagnosis of bovine paratuberculosis.
Insights
Researchers identified potential diagnostic proteins for bovine paratuberculosis. Natural forms of these proteins, particularly ModD, show promise for diagnosing bovine paratuberculosis, outperforming recombinant versions in ELISA tests.
Area of Science:
- Veterinary Immunology
- Bacteriology
- Protein Biochemistry
Background:
- Bovine paratuberculosis (Johne's disease) is a chronic infectious disease in cattle.
- Accurate diagnostics are crucial for controlling its spread.
- Previous studies identified 14 potential diagnostic proteins from Mycobacterium paratuberculosis.
Purpose of the Study:
- To express 14 identified open reading frames (ORFs) in E. coli.
- To evaluate the antigenicity and diagnostic potential of these expressed proteins.
- To compare the diagnostic performance of recombinant proteins versus natural antigens.
Main Methods:
- Expression of 14 ORFs in E. coli BL21(DE3) using pET-22b(+) vector.
- Purification of expressed proteins and confirmation of identity using polyclonal rabbit antibodies.
- Enzyme-linked immunosorbent assay (ELISA) using purified proteins (rModD) and natural antigens (ModD) with bovine sera.
- Receiver Operating Curve (ROC) analysis to assess diagnostic sensitivity and specificity.
Main Results:
- All 14 proteins were successfully expressed in E. coli, with higher yields of insoluble proteins.
- Five proteins (ModD, Antigen 85C, PepA, MAP1693c, MAP2168c) were confirmed as culture filtrate components.
- Recombinant ModD (rModD) showed reactivity with sera from infected cattle but did not improve ELISA sensitivity/specificity over crude antigens.
- Natural ModD demonstrated higher sensitivity and Area Under the Curve (AUC) in ELISA compared to rModD.
Conclusions:
- The natural forms of the identified proteins hold potential for diagnosing bovine paratuberculosis.
- Recombinant protein expression may lead to diminished antigenicity, impacting diagnostic utility.
- Further research into natural protein forms could lead to improved diagnostic tools for bovine paratuberculosis.
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