SDS-PAGE and Western blot analysis of somatic and extracellular antigens of Corynebacterium pseudotuberculosis

Abstract

Different techniques to isolate secreted, cell-surface located and somatic fractions of Corynebacterium pseudotuberculosis have been studied by SDS-PAGE and immunoblotting, by using a pool of sera from naturally infected goats. The secreted antigens were obtained from culture supernatant of the bacteria grown in a chemically defined medium or in Brain Heart Infusion medium. The surface fraction was obtained by NaCl 1 M treatment and the somatic ones were obtained by using distinct procedures (detergents and ultrasounds). By Coomassie blue staining, we observed 20 bands in the secreted fraction, 35 bands in the surface extract and 40 to 50 bands in the somatic fraction, depending on the extraction procedure. Sixteen immunoreactive proteins were detected among the studied fractions. Bands with molecular weights of 125, 108, 75, 68, 41, 40, 31 and 24 kDa were recognized with higher intensity and all of them were found in the secreted fraction. The use of the chemically defined medium allowed us to evidence the presence of proteins with high molecular masses in the secreted fraction of C. pseudotuberculosis, which had not been previously described.