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Cloning, nucleotide sequence, and expression of the Brucella melitensis bp26 gene coding for a protein immunogenic in
A Cloeckaert1, H S Debbarh, N Vizcaíno
1Institut National de la Recherche Agronomique, Laboratoire de Pathologie Infectieuse et Immunologie, Nouzilly, France. axel.cloeckaert@tours.inra.fr
Abstract:
We have previously identified a Brucella melitensis 28 kDa cytosoluble protein (CP28) which was highly immunogenic in infected sheep and which in addition made possible the serological differentiation between infected and B. melitensis Rev. 1 vaccinated sheep. Monoclonal antibodies against CP28 were used to screen a B. melitensis 16M genomic library and to clone the corresponding gene. DNA sequencing of the gene encoding CP28 of B. melitensis 16M revealed that it was nearly identical to that of the recently published bp26 gene of Brucella abortus vaccine strain S19 coding for a periplasmic protein. The differences between the B. melitensis 16M gene and that of B. abortus S19 consisted of single nucleotide substitutions, one or two codon deletions, one codon addition, and most importantly a 21-bp deletion. The corresponding region of B. abortus S19 contains two 10-bp direct repeats which could have been involved in the genesis of the deletion. Expression of the B. melitensis 16M bp26 gene in Escherichia coli studied by the use of the monoclonal antibodies showed the same characteristics as reported for the B. abortus S19 bp26 gene, i.e. the presence of a higher molecular mass preprotein and a lower molecular mass band which probably corresponds to the mature protein exported to the periplasm. Immunoblotting performed with sera from either naturally infected or B. melitensis H38 experimentally infected sheep confirmed the importance of the B. melitensis CP28/BP26 protein as diagnostic antigen.
Insights
Brucella melitensis CP28, a diagnostic antigen, shares high similarity with Brucella abortus BP26. Genetic differences, including a significant deletion, were identified between these Brucella species proteins.
Area of Science:
- Veterinary Microbiology
- Molecular Biology
- Immunology
Background:
- A previously identified Brucella melitensis 28 kDa cytosoluble protein (CP28) is highly immunogenic in sheep.
- CP28 facilitates serological differentiation between infected and vaccinated sheep.
Purpose of the Study:
- To clone and sequence the gene encoding B. melitensis CP28.
- To compare the B. melitensis CP28 gene with the Brucella abortus BP26 gene.
- To confirm the diagnostic utility of the B. melitensis CP28/BP26 protein.
Main Methods:
- Screening of a B. melitensis genomic library using monoclonal antibodies against CP28.
- DNA sequencing of the cloned gene.
- Gene expression studies in Escherichia coli.
- Immunoblotting assays with sheep sera.
Main Results:
- The B. melitensis CP28 gene is nearly identical to the B. abortus BP26 gene.
- Key differences include single nucleotide substitutions and a 21-bp deletion in the B. melitensis gene, potentially linked to direct repeats in B. abortus S19.
- Expressed B. melitensis CP28/BP26 protein exhibited characteristics similar to B. abortus BP26, including preprotein and mature forms.
- Immunoblotting confirmed CP28/BP26 as a crucial diagnostic antigen for Brucella infection in sheep.
Conclusions:
- The Brucella melitensis CP28 protein is homologous to Brucella abortus BP26, with notable genetic variations.
- The CP28/BP26 protein is a valuable diagnostic antigen for identifying Brucella infections in sheep.