IDENTIFICATION AND CHARACTERIZATION OF BRUCELLA-OVIS IMMUNOGENIC PROTEINS USING 2-DIMENSIONAL ELECTROPHORESIS AND IMMUNOBLOTTING

In a previous report, proteins from Brucella melitensis were character ized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and N-terminal microsequencing. In the present report, we have extende d this study to the second etiologic agent in ovine brucellosis, B. ov is, responsible far ram epididymitis and infertility. The combination of 2-D gel electrophoresis and protein microsequencing facilitated the location and identification of the major proteins of B. ovis on the 2 -D pattern. These proteins comprised cytoplasmic, periplasmic, and som e membrane proteins except the major outer membrane proteins. By compa ring 2-D gel profiles of B. ovis with that of B. melitensis described previously, a few proteins with different expression levels were readi ly identified. Serum from a ram naturally infected with B. ovis was us ed in immunoblotting studies to identify immunogenic proteins recogniz ed during the course of infection. This serum showed antibody reactivi ty against approximately 82 protein spots. Twenty-one of these protein s were identified either by use of monoclonal antibodies or by N-termi nal microsequencing. Several proteins previously described in earlier Brucella works were identified: the 89 kDa outer membrane protein, Dna K, GroEL, BP26, and Cu-Zn superoxide dismutase. Eight proteins had ami no acid sequences homologous to those of various proteins from other b acteria found in protein databases: NikA, dihydrolipoamide succinyltra nsferase, a hypothetical 31 kDa protein, malate dehydrogenase, succiny l-CoA synthetase alpha subunit, an amino acid ABC type transporter, Le u/Ile/Val-binding protein precursor, and CLpP. The remaining eight pro teins had N-terminal sequences lacking similarity to existing database entries. Thus, the 2-D PAGE analysis provided a convenient first appr oach in the characterization of immunogenic proteins.