CLONING, NUCLEOTIDE-SEQUENCE, AND EXPRESSION OF THE BRUCELLA-MELITENSIS BP26 GENE CODING FOR A PROTEIN IMMUNOGENIC IN INFECTED SHEEP

We have previously identified a Brucella melitensis 28 kDa cytosoluble protein (CP28) which was highly immunogenic in infected sheep and whi ch in addition made possible the serological differentiation between i nfected and B. melitensis Rev.1 vaccinated sheep. Monoclonal antibodie s against CP28 were used to screen a B. melitensis 16M genomic library and to clone the corresponding gene, DNA sequencing of the gene encod ing 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 t he B. melitensis 16M gene and that of B. abortus S19 consisted of sing le nucleotide substitutions, one or two codon deletions, one codon add ition, 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. mel itensis 16M bp26 gene in Escherichia coli studied by the use of the mo noclonal antibodies showed the same characteristics as reported for th e B. abortus S19 bp26 gene, i.e. the presence of a higher molecular ma ss preprotein and a lower molecular mass band which probably correspon ds to the mature protein exported to the periplasm. Immunoblotting per formed with sera from either naturally infected or B. melitensis H38 e xperimentally infected sheep confirmed the importance of the B. melite nsis CP28/BP26 protein as diagnostic antigen.