结核分支杆菌rpoB基因克隆与酶谱分析

Cloning and mapping of the rpoB gene in M. tuberculosis

目的克隆结核分支杆菌ｒｐｏＢ基因。方法以结核分支杆菌ｒｐｏＢ基因保守区的聚合酶链反应（ＰＣＲ）扩增片段为探针从已建立的结核分支杆菌基因文库中筛选ｒｐｏＢ基因。结果引物ＴＲ１、ＴＲ２ｂ扩增Ｈ３７ＲａＤＮＡ获１约４１１ｂｐ片段。经克隆及序列分析表明该片段与结核分支杆菌ｒｐｏＢ基因中央区域的序列同源。对约１２０００个克隆进行筛选，共获７个可疑阳性克隆，经过再次杂交证实其中３个克隆为阳性。其中１个克隆携带约３．８ｋｂ插入片段。一系列内切酶酶切分析后得出该３．８ｋｂ克隆片段的部分限制性内切酶酶谱。进一步分析表明所用４１１ｂｐ探针同源序列距两端分别为１ｋｂ和２．８ｋｂ。结论与报道的结核分支杆菌ｒｐｏＢ基因开放阅读框架比较，我们克隆的３．８ｋｂ片段包含结核分支杆菌ｒｐｏＢ完整基因的大部分。

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 H 37 Ra DNA. The cloning and sequencing results indicated that the 411 bp products were homogeneous to M. tuberculsis 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.