Immunological screening of a genomic M. bovis BCG library expressed in M. smegmatis and identification of the M. bovis BCG analog of ClpB.
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.
Division of Infectious Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4984, USA.
M
M
Bona
R
R
Nayak
M
M
Wu
M
M
Mincek
J J
JJ
Ellner
D000818:Animals; D000917:Antibody Formation; D000942:Antigens, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D015153:Blotting, Western; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000276:immunology; D029968:Escherichia coli Proteins; D015723:Gene Library; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics* / Q000276:immunology*; D009163:Mycobacterium bovis / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D014397:Tuberculosis, Pulmonary / Q000276:immunology
D000942:Antigens, Bacterial; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D049071:Endopeptidase Clp; C069775:ClpB protein, E coli