Use of monoclonal antibodies to facilitate identification, cloning, and purification of Chlamydia trachomatis hsp10.
As a requisite for a physiological and immunological investigation, reagents were developed that facilitated the identification and purification of Chlamydia trachomatis hsp10 (chsp10). Monoclonal antibodies that specifically recognize chsp10 were generated with multiple-antigen peptides (MAPs) to promote recognition of Chlamydia-specific epitopes. MAP2, containing amino acids 54 to 69 of the hsp10 sequence, elicited strong antibody responses after immunization of BALB/c mice. Monoclonal antibodies from several cloned hybridomas reacted on immunoblots with an approximately 15-kDa chlamydial protein and recombinant chsp10. Because of its strict specificity for chsp10, monoclonal antibody M1.2 was selected for routine use. M1.2 reacted by immunoblot with the hsp10s of several C. trachomatis strains but not with Chlamydia psittaci hsp10 or Escherichia coli homolog GroES, suggesting that M1.2 recognizes a species-specific epitope. Recombinant chsp10 was purified by immunoaffinity chromatography with M1.2. For large-scale purification, chsp10 was appended with a C-terminal six-histidine tag for purification by nickel chelate affinity chromatography. The hypA gene encoding the chsp10 of C. trachomatis serovar E/Bour was cloned into the pQE-60 vector (QIAGEN, Inc.) following PCR amplification from genomic DNA. E. coli DH5 transformants were screened for chsp10 expression by colony immunoblotting with M1.2, were tested for nickel matrix binding, and were sequenced. The sequence of serovar E/Bour chsp10 was found to be closely homologous to those of hsp10s of other chlamydiae. Purified chsp10 and specific anti-chsp10 monoclonal antibodies will be useful for investigating the biological and immunological roles of hsp10 in chlamydial infections.
LaVerda
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Department of Medical Microbiology and Immunology, University of Wisconsin, Madison 53706, USA.
Byrne
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GI
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015373:Bacterial Typing Techniques; D002692:Chlamydia trachomatis / Q000145:classification* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016415:Sequence Alignment
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal