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Identification and characterization of Brucella ovis immunogenic proteins using two-dimensional electrophoresis and immunoblotting.
- Source :
-
Electrophoresis [Electrophoresis] 1997 Aug; Vol. 18 (8), pp. 1491-7. - Publication Year :
- 1997
-
Abstract
- In a previous report, proteins from Brucella melitensis were characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and N-terminal microsequencing. In the present report, we have extended this study to the second etiologic agent in ovine brucellosis, B. ovis, responsible for 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 some membrane proteins except the major outer membrane proteins. By comparing 2-D gel profiles of B. ovis with that of B. melitensis described previously, a few proteins with different expression levels were readily identified. Serum from a ram naturally infected with B. ovis was used in immunoblotting studies to identify immunogenic proteins recognized during the course of infection. This serum showed antibody reactivity against approximately 82 protein spots. Twenty-one of these proteins were identified either by use of monoclonal antibodies or by N-terminal microsequencing. Several proteins previously described in earlier Brucella works were identified: the 89 kDa outer membrane protein, DnaK, GroEL, BP26, and Cu-Zn superoxide dismutase. Eight proteins had amino acid sequences homologous to those of various proteins from other bacteria found in protein databases; NikA, dihydrolipoamide succinyltransferase, a hypothetical 31 kDa protein, malate dehydrogenase, succinyl-CoA synthetase alpha subunit, an amino acid ABC type transporter, Leu/Ile/Val-binding protein precursor, and ClpP. The remaining eight proteins had N-terminal sequences lacking similarity to existing databases entries. Thus, the 2-D PAGE analysis provided a convenient first approach in the characterization of immunogenic proteins.
- Subjects :
- Amino Acid Sequence
Animals
Antibodies, Bacterial blood
Antibodies, Monoclonal
Antigens, Bacterial genetics
Antigens, Bacterial isolation & purification
Bacterial Proteins genetics
Bacterial Proteins immunology
Brucella genetics
Brucella immunology
Brucella melitensis chemistry
Brucella melitensis genetics
Brucella melitensis immunology
Brucellosis immunology
Brucellosis veterinary
Male
Molecular Sequence Data
Peptide Mapping methods
Sheep
Sheep Diseases immunology
Species Specificity
Bacterial Proteins isolation & purification
Blotting, Western methods
Brucella chemistry
Electrophoresis, Gel, Two-Dimensional methods
Subjects
Details
- Language :
- English
- ISSN :
- 0173-0835
- Volume :
- 18
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Electrophoresis
- Publication Type :
- Academic Journal
- Accession number :
- 9298663
- Full Text :
- https://doi.org/10.1002/elps.1150180824