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Cloning of a Brucella melitensis group 3 antigen gene encoding Omp28, a protein recognized by the humoral immune
L E Lindler1, T L Hadfield, B D Tall
1Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA.
Abstract:
Brucella group 3 antigens (Ags) are outer membrane proteins (OMPs) with a molecular mass ranging from 25 to 30 kDa. The OMPs are of interest partially because of their potential use as vaccine and diagnostic reagents. We used human convalescent antibody (Ab) to clone a gene that encoded a 28-kDa protein from a lambdagt11 library of Brucella melitensis 16M genomic DNA. DNA sequence analysis revealed a single open reading frame that would encode a protein of 26,552 Da. The 28-kDa protein had a primary amino acid sequence that was 43% similar to a previously described Brucella abortus group 3 Ag, Omp25 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The similarity to a known group 3 OMP, immunoreactivity with Ab prepared against B. abortus group Ags, immunolabeling of whole cells, and Southern hybridization led to our conclusion that the B. melitensis 28-kDa protein was a group 3 protein distinct from B. abortus Omp25. We designated the B. melitensis protein Omp28. Human convalescent sera from patients infected with B. abortus and Brucella suis as well as rabbit antisera prepared against killed B. abortus whole cells recognized B. melitensis Omp28 on Western blots (immunoblots). Furthermore, mice and goats infected with smooth strains of B. melitensis produced Abs against Omp28. Our results may begin to explain the variability in molecular weight seen in Brucella group Ags and point toward their possible use in vaccination against infection as well as diagnosis of the disease.
Insights
Brucella group 3 antigens, outer membrane proteins (OMPs), were identified as Omp28 in Brucella melitensis. This protein shows potential for use in vaccines and diagnostics for brucellosis.
Area of Science:
- Microbiology
- Immunology
- Protein Chemistry
Background:
- Brucella group 3 antigens are outer membrane proteins (OMPs) with potential as vaccine and diagnostic reagents.
- Variability in molecular weight of Brucella group antigens is observed.
Purpose of the Study:
- To clone and characterize a Brucella melitensis group 3 antigen.
- To investigate the potential of this antigen for diagnostic and vaccine development.
Main Methods:
- Cloning of a Brucella melitensis gene encoding a 28-kDa protein using human convalescent antibody.
- DNA sequence analysis to determine the protein's open reading frame and molecular mass.
- Sequence similarity comparison, Western blotting, immunolabeling, and Southern hybridization to characterize the protein.
Main Results:
- A 28-kDa Brucella melitensis outer membrane protein, designated Omp28, was identified and characterized.
- Omp28 shares 43% sequence similarity with Brucella abortus Omp25 but is distinct.
- Omp28 was recognized by human convalescent sera from Brucella-infected patients and by antisera against B. abortus.
- Infection with B. melitensis induced antibodies against Omp28 in mice and goats.
Conclusions:
- The identified Brucella melitensis 28-kDa protein is a distinct group 3 antigen, Omp28.
- Omp28 holds promise as a valuable reagent for the diagnosis and vaccination against brucellosis.