title: Sequence and immunogenicity of the 70-kDa heat shock protein of Mycobacterium leprae.
abstract: The gene encoding the Mycobacterium leprae 70-kDa heat shock protein has been isolated from a cosmid library using a fragment of the clone JKL2. Southern blot analysis of a positive clone identified a 4.4-kb fragment containing the entire coding region of the gene plus 2.4 kb upstream. Sequencing revealed the gene to encode a 621-amino acid protein, bearing 56% identity with the Escherichia coli dnaK gene product and 47% and 46% identity with the human and Caenorhabditis elegans hsp70, respectively. Comparison with the C-terminal 203 amino acids of the Mycobacterium tuberculosis 71-kDa Ag yielded 70% identity. Recombinant M. leprae p70 was produced in E. coli as a fusion protein (rp70f) with a portion of the schistosomal glutathione-S-transferase, using the expression vector, pGEX-2T. Cleavage with thrombin resulted in the release of a 70.0-kDa protein (rp70c) from the glutathione-S-transferase. Examination of the proteins by immunoblotting demonstrated that anti-M. leprae mAb, L7, and sera from lepromatous leprosy patients bound to both the cleaved and fusion proteins. We compared the T cell reactivity of the M. leprae recombinant proteins with that of mAb affinity-purified bacille Calmette-Guerin (BCG) 70-kDa Ag using proliferation assays. PBMC of BCG vaccinees responded to both M. leprae cleaved and fusion p70, though more subjects responded to the rp70c (18 of 20) than to rp70f (13 of 20). Responses were generally higher to rp70c than to rp70f, however all responses to the M. leprae recombinant proteins were lower than to mAb affinity-purified BCG p70. Thus, the M. leprae 70-kDa heat shock protein elicits T and B cell responses in subjects exposed to mycobacteria, despite its homology with the human hsp70.
authors.affiliation: Centenary Institute of Cancer Medicine and Cell Biology, University of Sydney, Australia.
authors.forename: K R
authors.initials: KR
authors.lastname: McKenzie
authors.forename: E
authors.initials: E
authors.lastname: Adams
authors.forename: W J
authors.initials: WJ
authors.lastname: Britton
authors.forename: R J
authors.initials: RJ
authors.lastname: Garsia
authors.forename: A
authors.initials: A
authors.lastname: Basten
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000276:immunology; D006801:Humans; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D009166:Mycobacterium leprae / Q000235:genetics* / Q000276:immunology; D015183:Restriction Mapping
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006360:Heat-Shock Proteins