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[Cloning and mapping of the rpoB gene in M. tuberculosis]
Objective:
To clone the rpoB gene of M. tuberculosis.
Methods:
Screening the rpoB gene of M. tuberculosis from M. tuberculosis genomic DNA library by using the rpoB gene conservative region PCR product as a probe.
Results:
411 bp products were seen after amplification of H37Ra DNA. The cloning and sequencing results indicated that the 411 bp products were homogeneous to M. tuberculosis rpoB gene. 12,000 clones of M. tuberculosis genomic DNA library were screened by hybridization using the 411 bp product as a probe and 7 clones seemed positive, 3 clones were real positive after the second hybridization. 1 of them carried a 3.8 kb insert fragment. The preliminary restriction map of the 3.8 kb segment was made. The regions which were found homogeneous to the probe to the end of this cloned fragment were 1 and 2.8 kb.
Conclusion:
In comparison with the open reading frame of H37Rv rpoB gene, the 3.8 kb segment cloned here covers larger portion of entire rpoB gene of M. tuberculosis. This study will provide a useful tool to study the resistant mechanism of rifampin and other rifamycin compounds to M. tuberculosis.