Cloning, sequence determination, and expression of a 32-kilodalton-protein gene of Mycobacterium tuberculosis

Abstract

We describe the identification of the gene encoding an immunodominant 32-kilodalton (kDa) protein of Mycobacterium tuberculosis. The 32-kDa antigen is abundantly secreted into the culture supernatant of a variety of mycobacteria and appears to be a major stimulant of cellular and humoral immunity against mycobacteria. Recombinant clones expressing a 140or 125-kDa j8-galactosidase fusion protein reactive with rabbit polyclonal anti-32 kDa protein serum were detected. The corresponding DNA sequence contains a 1,008-base-pair coding region. The deduced amino acid sequence corresponds to a 336-residue protein including the previously determined NH2-terminal sequence of the 32-kDa protein (Microb. Pathog. 2:351-366, 1987). Upstream of this NH2-terminal region, the gene codes for a signal peptide required for the secretion of a 294-amino-acid-long mature protein. A putative promotor sequence could be located upstream of the open reading frame. Comparison of the M. tuberculosis 32-kDa antigen with the Mycobacterium bovis BCG a-antigen (K. 170:3847-3854, 1988) revealed 73.8% homology between DNA sequences and 72.8% homology between amino acid sequences (signal and mature protein). Finally, the 140-kDa fusion protein could selectively be recognized by human tuberculous sera. This result confirms our previous finding that the 32-kDa antigen could be a valuable tool for the serological diagnosis of tuberculosis. Moreover, the availability of recombinant proteins opens perspectives for the localization of relevant B-and T-cell epitope regions on the 32-kDa antigen.

Characterization of the Gene Encoding the MPB51, One of the Major Secreted Protein Antigens of Mycobacterium bovis BCG, and Identification of the Secreted Protein Closely Related to the Fibronectin Binding 85 Complex

“…The upstream region of the BCG 85A gene, from -274 to -251. was more A/T rich than ihe BCG 85A coding region (66.7% as opposed to 32.9%). Although Martine Borremans et al and Luk De Wit et al reported the sequence of 85A genes from M. tuberculosis and BCG, they did not contain this region [14,15]. The sequence of this region is similar to the E. coli CAP-binding site consensus sequence [40], The A/T rich regions were seen near the promoter region of several genes and were binding sites for transcription factors.…”

“…The analogous gene was cloned recently from Mycobacterium leprae [12,13]. The genes for the 85A component were cloned from M. tubercuhm.s [14]. BCG [15], and M. leprae [16].…”

TB vaccines: current status and future perspectives

“…[54][55][56][57] Some of the more prominent antigens found in this fraction included the three members of the Ag85 complex (Rv3804c, Rv1886c, Rv0129c), plus Rv1196/PPE18, Rv0125/PepA, Rv0915c/PPE14 and Rv3616c. 58,59 In the lower molecular mass fraction, several proteins belonging to the ESAT-6 family (early secretory antigenic target-6) including ESAT-6/Rv3875, CFP10/ Rv3874, TB9.8/Rv0287 and Mtb9.9A/Rv1793 were isolated. [60][61][62][63] The ESAT-6 family was identified after the completion of the M. tuberculosis genome-this family is not based on homology among the members (which is quite limited) but rather on their organization on the bacterial chromosome where they are found as adjacent pairs close to a PPE or a PE gene.…”

Vaccination strategies against intracellular microbes

“…Mycobacteria are Gram-positive bacteria, as is L. monocytogenes, but due to the highly complex, lipoid-rich cell wall structure of mycobacteria, the export machinery including signal-peptidase specificity may differ from L. monocytogenes. Yet, structural homology between BCG and Gram-positive bacterial signal peptide sequences has been reported [58,59]. The S. typhimurium aroA-strain belongs to the Gram-negative world as does E. coli.…”