Cloning, expression, purification and serodiagnostic evaluation of fourteen Mycobacterium paratuberculosis proteins

Donghee Cho, Sung Jae Shin, Adel M. Talaat, Michael T. Collins

Research output: Contribution to journalArticle

36 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)411-420
Number of pages10
JournalProtein Expression and Purification
Volume53
Issue number2
DOIs
Publication statusPublished - 2007 Jun 1

All Science Journal Classification (ASJC) codes

  • Biotechnology

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