Construction of recombinant neisserial Hsp60 proteins and mapping of antigenic domains.
Here we report the cloning and expression, in Escherichia coli, of PCR-amplified DNA encoding the 63-kDa stress-inducible protein of Neisseria gonorrhoeae strains VP1 and PID2, Neisseria meningitidis 2996 and the commensal Neisseria flavescens. DNA sequence analysis revealed in all cases one open reading frame of 541-544 amino acids corresponding to a protein of approximately 57,000 Da. The various neisserial proteins were > 96% identical at the amino acid level and showed extensive homology with proteins belonging to the Hsp60 heat-shock-protein family. We constructed defined glutathione S-transferase fusion polypeptides of the gonococcal Hsp60 homologue to locate antigenic domains on the recombinant protein. Variation in the immunoreactivity of two monoclonal antibodies recognizing a conserved and a neisseria-unique antigenic Hsp60 determinant, respectively, could thus be deduced to result from single amino acid substitutions. Analysis of the antibody response in patients' sera demonstrated reactivity with the same fusion polypeptides in six out of nine sera, indicating that neisserial Hsp60 is expressed during the natural infection and that distinct domains on the protein are immunodominant in vivo.
Pannekoek
Y
Y
Max-Planck-Institut für Biologie, Abteilung Infektionsbiologie, Tübingen, Germany.
Dankert
J
J
van Putten
J P
JP
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics* / Q000276:immunology; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D016056:Immunodominant Epitopes / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D009343:Neisseria / Q000235:genetics* / Q000276:immunology; D009344:Neisseria gonorrhoeae / Q000235:genetics / Q000276:immunology; D009345:Neisseria meningitidis / Q000235:genetics / Q000276:immunology; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D016415:Sequence Alignment
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D018834:Chaperonin 60; D004269:DNA, Bacterial; D016056:Immunodominant Epitopes; D011993:Recombinant Fusion Proteins