A 20.9kDa novel putative lipoprotein from Mycobacterium paratuberculosis is strongly antigenic

GIOFFRÉ A.; BIGI F.; ALITO A.; CAIMI K.; SANTANGELO M.; ZUMÁRRAGA M.; MORETTA R.; MEIKLE V.; PAOLICCHI F.; CATALDI A.; ROMANO M.

Bariloche, Río Negro

Congreso; XXXVII Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2003

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Mycobacterium avium subsp. paratuberculosis (MPTB) is an important cause of ruminant animal disease such as paratuberculosis. The sequence of MPTB genome has recently been finished however a large proportion of the putative genes still have no identified functions. To identify a novel potential diagnostic antigen molecule we analyzed an expression library with serum of animals naturally infected with the bacteria or immunized mice. We identified an ORF of 611 pb that codes a hypothetical protein of 20872 MW. The sequence has 99% of identity with the corresponding ORF from de genome of M.avium subsp. avium (BLAST search). The presence of the gene was evaluated by Southern Blot and PCR in other species of mycobacterium genera. The results demonstrate that this sequence was only presents in these species and as well in M. plhei. Database of protein families and domains searching (PROSITE) revealed that the protein could have prokaryotic membrane lipoprotein lipid attachment site. The localisation to the bacterial membrane is hypothesized. The ORF was cloned an expressed in pRSET vector, and a recombinant peptide of 32.7kDa was obtained and purified. Mice were immunized to obtain policlonal sera to perform cellular localization. Humoral response was evaluated in sera and milk serum samples from different animals (bovine, ovine) by immunoblot, demonstrating that this protein is strongly recognized.