Molecular Cloning of the Gene for a Conserved Major Immunoreactive 28-Kilodalton Protein ofEhrlichia canis: a Potential Serodiagnostic Antigen

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Abstract
A gene encoding a 28-kDa protein ofEhrlichia caniswas cloned, sequenced, and expressed, and a comparative molecular analysis with homologous genes ofE. canis,Cowdria ruminantium, andEhrlichia chaffeensiswas performed. The 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 signal sequence was identified, suggesting that the protein undergoes posttranslational modification to a mature 27.7-kDa protein (P28). TheE. canis p28gene has significant nucleic acid and amino acid sequence homologies with theE. chaffeensisouter membrane protein-1 (omp-1) gene family, with theCowdria ruminantium map-1gene, and with otherE. canis28-kDa-protein genes. Southern blotting revealed the presence of at least two additional homologousp28gene copies in theE. canisgenome, confirming thatp28is a member of a polymorphic multiple-gene family. Amino acid sequence analysis revealed thatE. canisP28 has four variable regions, and it shares similar surface-exposed regions, antigenicity, and T-cell motifs withE. chaffeensisP28. Thep28genes from seven differentE. canisisolates were identical, indicating that the gene for this major immunoreactive protein is highly conserved. In addition, reactivity of sera from clinical cases of canine ehrlichiosis with the recombinant P28 demonstrated that the recombinant protein may be a reliable serodiagnostic antigen.