Immunological screening of a genomic M. bovis BCG library expressed in M. smegmatis and identification of the M. bovis BCG analog of ClpB

• Published in: Tubercle and lung disease 1998-01, Vol.78 (2), p.133-141
• Main Authors: Bona, M., Nayak, R., Wu, M., Mincek, M., Ellner, J.J.
• Format: Article
• Language: English
• Subjects: Animals; Antibody Formation; Antigens, Bacterial - genetics; Antigens, Bacterial - isolation & purification; Base Sequence; Blotting, Western; Endopeptidase Clp; Escherichia coli - immunology; Escherichia coli Proteins; Gene Library; Heat-Shock Proteins - genetics; Humans; Molecular Sequence Data; Mycobacterium - genetics; Mycobacterium - immunology; Mycobacterium bovis - genetics; Mycobacterium bovis - immunology; Rabbits; Tuberculosis, Pulmonary - immunology
• ISSN: 0962-8479; 1532-219X
• DOI: 10.1016/S0962-8479(98)80006-3

This work describes the screening of a M. bovis BCG cosmid library in M. smegmatis with a hyperimmune rabbit anti-BCG serum. Cross-reactive antibodies interfere with the detection of BCG specific antigens in M. smegmatis culture filtrates. We, therefore, screened parallel western blots with serum adsorbed with a M. smegmatis cell lysate and unadsorbed serum. Comparison of the western blots allowed distinction between BCG specific and cross-reactive M. smegmatis antigens. Thirty-one cosmids expressed BCG specific antigens. One of them, a hitherto undescribed 100 kDa antigen was subcloned, sequenced and expressed in E. coli. It shows a high degree of homology to ClpB, a member of the Clp family of proteases and was immunologically reactive with the rabbit hyperimmune serum against M. bovis BCG. A positive signal was also obtained with sera of patients with tuberculosis. This antigen is a previously unrecognized target of the human immune response to mycobacteria.