CONSTRUCTION OF RECOMBINANT NEISSERIAL HSP60 PROTEINS AND MAPPING OF ANTIGENIC DOMAINS

Here we report the cloning and expression, in Escherichia coil, of PCR -amplified DNA encoding the 63-kDa stress-inducible protein of Neisser ia gonorrhoeae strains VP1 and PID2, Neisseria meningitidis 2996 and t he commensal Neisseria flavescens. DNA sequence analysis revealed in a ll cases one open reading frame of 541-544 amino acids corresponding t o a protein of approximately 57 000 Da. The various neisserial protein s were >96% identical at the amino acid level and showed extensive hom ology with proteins belonging to the Hsp60 heat-shock-protein family. We constructed defined glutathione S-transferase fusion polypeptides o f the gonococcal Hsp60 homologue to locate antigenic domains on the re combinant protein. Variation in the immunoreactivity of two monoclonal antibodies recognizing a conserved and a neisseria-unique antigenic H sp60 determinant, respectively, could thus be deduced to result from s ingle amino acid substitutions. Analysis of the antibody response in p atients' sera demonstrated reactivity with the same fusion polypeptide s in six out of nine sera, indicating that neisserial Hsp60 is express ed during the natural infection and that distinct domains on the prote in are immunodominant in vivo.