Cloning and expression of mycobacterial genes in Escherichia coli.

The ability of Escherichia coli to use the expression signals of mycobacterial genes was tested by inserting fragments of M. bovis BCG DNA into the E. coli promoter-probe plasmid pKK232-8. Comparison with the promoter activity achieved following insertion of restriction fragments of the E. coli host into. pKK232-8 revealed that a significant proportion of M. bovis BCG promoters were functional in E. coli. These results confirmed the suitability of E. coli as a host for the cloning and expression of mycobacterial genes. Using a variety of E. coli cloning vectors (pBR322, pUC13, EMBL4 and gtll), M. bovis BCG and M. leprae DM gene libraries were prepared. Recombinant M. bovis BCG clones were screened with rabbit antiserum and clones expressing M. bovis BCG antigens were identified. A pBR322/M. bovis BCG clone, expressing a 65KD molecule, was isolated and this antigen was shown to be cross-reactive with a 65KD M. leprae antigen. Recombinant gtll clones, expressing antigenic M. bovis BCG molecules, were also detected and a partial DM sequence was determined for one of these molecules. Moreover, recombinant gtll clones expressing (i) an 85KD biotinylated M. leprae molecule and (ii) an 85KD biotinylated M. bovis BCG molecule were also detected. In an attempt to test the feasibility of diagnosing leprosy by the presence of antibodies to specific antigens, antisera samples from leprosy patients and their contacts were screened for antibodies to mycobacterial antigens. Although only a small number of antisera were tested, a number of candidate antigens were identified