title: Molecular cloning of a gene encoding the immunogenic 21 kDa protein of Cowdria ruminantium.
abstract: Major immunogenic polypeptides (21, 32, 40, 46, 58, 85 and 160 kDa) of Cowdria ruminantium were identified by immunoprecipitation and immunoblotting. A pUC13 library of C. ruminantium genomic DNA was screened with hyperimmune sheep serum to identify Escherichia coli colonies which expressed genes encoding these immunogenic proteins. A recombinant E. coli colony, F5.2, was identified containing plasmid insert DNA of 2773 bp. The cloned DNA insert contained two long open reading frames (ORFs) of 627 bp (complete) and 831 bp (incomplete), both potentially encoding proteins containing an N-terminal signal peptide. Deletion experiments suggested that the hyperimmune sheep serum recognized a protein that was encoded by the 627 bp ORF. The 627 bp ORF was amplified by polymerase chain reaction (PCR), subcloned and expressed to a high level in E. coli. A sheep antiserum made to the expressed recombinant fusion protein recognized a 21 kDa protein of all strains of C. ruminantium tested, confirming that the 627 bp ORF encodes a native 21 kDa protein in C. ruminantium. Similarly, the recombinant protein was recognized by all sera tested from heartwater-infected animals. The antigenic conservation of the 21 kDa protein and its immunogenic nature are reasons for further testing of this recombinant protein in subunit diagnostic tests.
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville 32611-0880.
authors.forename: S M
authors.initials: SM
authors.lastname: Mahan
authors.forename: T C
authors.initials: TC
authors.lastname: McGuire
authors.forename: S M
authors.initials: SM
authors.lastname: Semu
authors.forename: M V
authors.initials: MV
authors.lastname: Bowie
authors.forename: F
authors.initials: F
authors.lastname: Jongejan
authors.forename: F R
authors.initials: FR
authors.lastname: Rurangirwa
authors.forename: A F
authors.initials: AF
authors.lastname: Barbet
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D016995:Ehrlichia ruminantium / Q000235:genetics* / Q000276:immunology; D005798:Genes, Bacterial; D006357:Heartwater Disease / Q000382:microbiology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D015030:Zimbabwe
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C086402:major antigenic protein 1, Cowdria ruminantium