A rabbit polyclonal antiserum exhibiting a specific recognition pattern for Mycobacterium tuberculosis proteins was used to screen an M. tuberculosis genomic library constructed in the expression vector lambda gtll. One clone, denominated C1:10, expressed M. tuberculosis-specific determinants as part of a large fusion protein with ,I-galactosidase. The gene for this protein has been sequenced, and it encodes a protein of 134 amino acids (13.8 kDa) which did not display significant homology with any of the previously reported proteins in the data bases. Hybridization studies with restriction fragments of the cloned sequence revealed that it was not present in the genomes of related mycobacteria, namely, M. bovis, M. bovis BCG, M. flavescens, M. fortuitum, M. phlei, and M. vaccae. These findings suggest that we have detected a gene, or a fragment therefrom, unique for M. tuberculosis whose nucleotide and amino acid sequences could be useful tools in the design of an improved vaccine or a diagnostic method of greater accuracy for tuberculosis. * Corresponding author. focused our studies on those proteins exclusively present in M. tuberculosis and not in M. bovis BCG. Here, we describe the results obtained when TB40 serum, a rabbit serum raised against one of these M. tuberculosis-specific proteins, was used to screen a lambda gtll genomic library. MATERIALS AND METHODS Bacterial strains and vectors. The following mycobacterial strains were obtained from the Trudeau Mycobacterial Collection (TMC): M. tuberculosis (TMC 102, strain H37Rv), M. bovis (TMC 410), M. bovis BCG (TMC 1011, substrain Pasteur), M. phlei (ATCC 11758), M. vaccae (TMC 1526), M. flavescens (ATCC 14474), and M. fortuitum (TMC 1529). Bacteriophage lambda gtll and Escherichia coli Y1088, Y1089, and Y1090 were provided by Amersham (Amersham, United Kingdom). E. coli DH5a, E. coli XL1-Blue, and phagemid vector Bluescript were purchased from Stratagene (La Jolla, Calif.). M13mpl8 was from Pharmacia (Uppsala, Sweden). Sonic extracts. Mycobacteria were grown on Sauton's medium, harvested, and sonicated as described by Janicki et al. (16) with minor modifications. Briefly, the bacilli were sonicated by subjecting them to 15-min pulses in a Branson sonicator at 0°C, followed by a 5-min rest period between pulses, and the process was repeated four times. The sonicate was then centrifuged at 150,000 x g for 1 h at 4°C. The supernatant was removed, and the protein content was determined by the method of Lowry et al. (20). This material was stored in aliquots at-70°C until needed. Antiserum and immunological analysis. Polyclonal antisera raised against purified M. tuberculosis proteins have been produced by us and described elsewhere (25). Briefly, 150 ,ug of the MTP40 protein, which had been isolated from polyacrylamide gels under nonreducing conditions, was mixed volume for volume with incomplete Freund adjuvant and injected subcutaneously into rabbits on days 0, 20, and 45. The rabbits were bled on days 30, 45, and 60. The antiserum obtained after this immuni...