Molecular cloning and expression of Mycobacterium tuberculosis strain Aoyama B peptide antigen genes in Escherichia coli--a gene encoding 15 kD antigen (AT 01)

A genomic library of Mycobacterium tuberculosis strain Aoyama B in Escherichia coli K-12 was constructed by cloning Sau 3A I cleaved M. tuberculosis Aoyama B chromosomal DNA into pUC18, pUC181 or pUC182. Clones reacting with anti-PPDs-rabbit-serum were screened by immunoblotting among 3 x 10(4) clones. Seven clones were selected; designating pAT 01, pAT 101, pAT 102, pAT 103, pAT 104, pAT 105 and pAT 201. On Western blotting, they were shown to produce 15 kD (pAT 01, pAT 101, pAT 105), 18 kD (pAT 103) and 60 kD (pAT 102, pAT 201) peptide antigens. Restriction endonuclease map of each of the above clones was composed, and putative coding frames of anti-PPDs-rabbit-serum reactive peptides were deduced by analysing deletion derivatives. Nucleotide sequence of pAT 01, encoding for 15 kD peptide antigen was analyzed, and a Hinf I-Hinf I fragment in pAT 01 was subcloned into pUC 18 lambda CPL 1 to determine the direction of its reading frame. The origin of promoter that drives cloned mycobacterial genes in E. coli was discussed.