Submitted to: Proteomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/23/2007
Publication Date: 2/1/2008
Citation: Bannantine, J.P., Waters, W.R., Stabel, J.R., Palmer, M.V., Li, L., Kapur, V., Paustian, M. 2008. Development and Use of a Partial Mycobacterium avium subspecies paratuberculosis Protein Array. Proteomics. 8(3):463-474. Interpretive Summary: This article reports on our very initial efforts at developing a protein array for M. paratuberculosis, the bacterium that causes Johne’s disease. We cloned a set of 43 genes from this genome and recombinant produced protein from each of these clones. The article carefully details how this was accomplished and lists some of the obstacles encountered with the difficult to express proteins. As a proof of concept, a protein array was constructed using a dot blotting device and nitrocellulose sheets, which served as the solid phase platform. The arrays were spotted and exposed to sera from Johne’s disease cattle as well as mice and rabbits that had been immunized with live M. paratuberculosis. Antigens were readily identified in all hosts examined and these studies provided motivation to continue with the time investment required to develop more comprehensive protein arrays.
Technical Abstract: As an initial step toward systematically characterizing all antigenic proteins produced by a significant veterinary pathogen, 43 recombinant Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) expression clones were constructed, cataloged and stored. Nitrocellulose filters were spotted with purified proteins from each clone along with a whole cell lysate of M. paratuberculosis. Spots on the resulting dot array consisted of hypothetical proteins (13), metabolic proteins (3), cell envelope proteins (7), known antigens (4) and unique proteins with no similarity in public sequence databases (16). Dot blot arrays were used to profile antibody responses in a rabbit and mouse exposed to M. paratuberculosis as well as in cattle showing clinical signs of Johne’s disease. The M. paratuberculosis heat shock protein DnaK, encoded by open reading frame MAP3840 and a membrane protein MAP2121c, were identified as the most strongly immunoreactive in both the mouse and rabbit hosts, respectively. Whereas MAP3155c, which encodes a hypothetical protein, was most strongly immunoreactive in Johne’s disease cattle. This study has enabled direct comparisons of antibody reactivity for an entire panel of over 40 proteins and laid the foundation for future high throughput production and arraying of M. paratuberculosis surface proteins for immune profiling experiments in cattle.