Characterization of two conformational epitopes of the Chlamydia trachomatis serovar L2 DnaK immunogen.
Chlamydia trachomatis DnaK is an important immunogen in chlamydial infections. DnaK is composed of a conserved N-terminal ATP-binding domain and a variable C-terminal peptide-binding domain. To locate the immunogenic part of C. trachomatis Dnak, we generated monoclonal antibodies (MAbs) against this protein. By use of recombinant DNA techniques, we located the epitopes for two MAbs in the C-terminal variable part. Although the antibodies reacted in an immunoblot assay, it was not possible to map the epitopes completely by use of 16-mer synthetic peptides displaced by one amino acid corresponding to the C-terminal part of C. trachomatis DnaK. To determine the limits of the epitopes, C. trachomatis DnaK and glutatione S-transferase fusion proteins were constructed and affinity purified. The purified DnaK fusion proteins were used for a fluid-phase inhibition enzyme-linked immunosorbent assay with the two antibodies. The epitopes were found not to overlap. To obtain DnaK fragments recognized by the antibodies with the same affinity as native C. trachomatis DnaK, it was necessary to express, respectively, regions of 127 and 77 amino acids. The MAbs described in this study thus recognized conformational epitopes of C. trachomatis DnaK.
Birkelund
S
S
Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark.
Mygind
P
P
Holm
A
A
Larsen
B
B
Beck
F
F
Christiansen
G
G
D000251:Adenosine Triphosphatases / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology*; D001483:Base Sequence; D002692:Chlamydia trachomatis / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli