RESUMO
To obtain recombinant peptides related to PPDs, we constructed a genomic library from the DNA of Mycobacterium tuberculosis Aoyama B, a standard strain in Japan to manufacture PPDs, using plasmid vectors pUC18 series. Seven clones reacting the anti-PPDs-rabbit-serum on immunoblotting were obtained, and restriction map was analysed. A nucleotide sequence and a putative open reading frame (ORF) of pAT01, encoding 15kD peptide, as well as the mode of expression was reported previously). In this study, nucleotide sequence of 60 kD peptide gene was determined, and the comparative database analysis (GENBANK) revealed a striking level of homology to so called mycobacterial heat shock protein. The expression mode of pAT201 encoding 60 kD, as well as pAT01 encoding 15 kD peptide, indicated that these peptides were not hybrid proteins with the lacZ gene product, but they were consisted of peptides only mycobacterial source. Therefore, 15 kD and 60 kD directly were subjected to immunological studies. The peptides were extracted from E. coli, carrying pAT01 or pAT201, purified through series of DEAE chromatography and followed by Detoxi-Gel to remove LPS. 15 kD peptide behaved almost similar to PPDs both in the DTH skin reaction and the lymphocyte proliferation response on guinea pigs or rats in respect to sensitivity. However, 60 kD was unique in that, and it behaved like a general mitogen. We discussed role of 60 kD peptide, comparing with the common antigen, generally found in most species of bacteria as the heat shock protein.
