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Cloning, nucleotide sequence, and expression of the Brucella melitensis omp31 gene coding for an immunogenic major
N Vizcaíno1, A Cloeckaert, M S Zygmunt
1Laboratoire de Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, Nouzilly, France. vizcaino@tours.inra.fr
Abstract:
The gene coding for the major outer membrane protein (OMP) of 31 to 34 kDa, now designated Omp31, of Brucella melitensis 16M was cloned and sequenced. A B. melitensis 16M genomic library was constructed in lambda GEM-12 XhoI half-site arms, and recombinant phages expressing omp31 were identified by using the anti-Omp31 monoclonal antibody (MAb) A59/10F09/G10. Subcloning of insert DNA from a positive phage into pGEM-7Zf allowed the selection of a plasmid bearing a 4.4-kb EcoRI fragment that seemed to contain the entire omp31 gene under control of its own promoter. omp31 was localized within a region of the EcoRI insert of approximately 1.1 kb. Sequencing of this region revealed an open reading frame of 720 bp encoding a protein of 240 amino acids and a predicted molecular mass of 25,307 Da. Cleavage of the first 19 amino acids, showing typical features of signal peptides for protein export, leaves a mature protein of 221 amino acids with a predicted molecular mass of 23,412 Da. The predicted amino acid sequence of B. melitensis 16M Omp31 showed 35.2% identity with the RopB OMP of Rhizobium leguminosarum bv. viciae 248 and 34.3% identity with Omp25 of B. abortus 544. As in Brucella spp., Omp31 was located in the outer membrane of recombinant Escherichia coli, but its reported peptidoglycan association in Brucella cells was not detected in E. coli. The ability of Omp31 to form oligomers resistant to sodium dodecyl sulfate denaturation at low temperatures, a characteristic described for several bacterial porins, was observed in both B. melitensis and recombinant E. coli. The epitope recognized by the anti-Omp31 MAb A59/10F09/G10, for which a protective activity has been suggested, has been delimited to a region of 36 amino acids of Omp31 covering the most hydrophilic part of the protein. The availability of recombinant Omp31 and the identification of the antigenic determinant recognized by MAb A59/10F09/G10 will allow the evaluation of their potential protective activity and their potential for the development of subcellular vaccines against brucellosis.
Insights
The Brucella melitensis Omp31 gene was cloned and sequenced, revealing its outer membrane protein structure and potential for vaccine development. This research identifies a key antigenic determinant for potential subcellular vaccines against brucellosis.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- Brucella melitensis is a significant pathogen causing brucellosis.
- Outer membrane proteins (OMPs) are crucial for bacterial structure and interaction.
- Omp31 is a major OMP in Brucella melitensis with potential antigenic properties.
Purpose of the Study:
- To clone, sequence, and characterize the gene encoding the major outer membrane protein (Omp31) of Brucella melitensis 16M.
- To investigate the structural and functional properties of Omp31.
- To identify antigenic determinants for potential vaccine development against brucellosis.
Main Methods:
- Construction and screening of a Brucella melitensis genomic library.
- Gene cloning, subcloning, and DNA sequencing.
- Expression of Omp31 in recombinant Escherichia coli.
- Analysis of protein sequence, localization, and oligomerization.
- Epitope mapping using monoclonal antibodies.
Main Results:
- The omp31 gene was cloned and sequenced, encoding a 240-amino acid protein.
- The mature Omp31 protein consists of 221 amino acids with a predicted mass of 23,412 Da.
- Omp31 showed sequence homology to other bacterial OMPs and was localized to the outer membrane.
- The protein formed SDS-resistant oligomers, characteristic of porins.
- A 36-amino acid hydrophilic region was identified as the epitope for a potentially protective monoclonal antibody.
Conclusions:
- The omp31 gene and its encoded protein have been characterized.
- Omp31 exhibits properties consistent with a porin and is located in the outer membrane.
- The identified epitope on Omp31 is a promising target for developing subcellular vaccines against brucellosis.