Molecular cloning of the gene for a conserved major immunoreactive 28-kilodalton protein of Ehrlichia canis: a potential serodiagnostic antigen

A gene encoding a 28-M)a protein of Ehrlichia canis was cloned, sequenced, and expressed, and a comparative molecular analysis with homologous genes o ff. canis, Cowdria ruminantium, and Ehrlichia chaffeensis was performed. Th e complete gene has an 834-bp open reading frame encoding a protein of 278 amino acids with a predicted molecular mass of 30.5 kDa. An N-terminal sign al sequence was Identified, suggesting that the protein undergoes posttrans lational modification to a mature 27.7-kDa protein (P28). The E. canis p28 gene has significant nucleic acid and amino acid sequence homologies with t he E. chaffeensis outer membrane protein-1 (omp-1) gene family, with the Co wdria ruminantium map-1 gene, and with other E. canis 28-kDa-protein genes. Southern blotting revealed the presence of at least two additional homolog ous p28 gene copies in the E. canis genome, confirming that p28 is a member of a polymorphic multiple-gene family. Amino acid sequence analysis reveal ed that E. canis P28 has four variable regions, and it shares similar surfa ce-exposed regions, antigenicity, and T-cell motifs with E. chaffeensis P28 . The p28 genes from seven different E. canis isolates were identical, indi cating that the gene for this major immunoreactive protein is highly conser ved. In addition, reactivity of sera from clinical cases of canine ehrlichi osis with the recombinant P28 demonstrated that the recombinant protein may be a reliable serodiagnostic antigen.