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【目的】通过分析结核分枝杆菌无毒株H37Ra的全基因组序列,并与H37Rv基因组序列比较,发现pabB和lpdA预测的启动子区发生了突变。我们利用报告基因,确认启动子突变与其基因转录水平的关系,探索结核分枝杆菌H37Ra毒力丧失的内在原因。【方法】利用生物信息学方法预测这两对基因的启动子区,采用PCR技术克隆这两对基因的启动子,与分枝杆菌启动子探针载体pMC210相连,DNA测序证实连接片段正确后,用电穿孔法将重组质粒转化至耻垢分枝杆菌mc2155。利用Quantitative Real-Time RT-PCR检测报告基因lacZ转录水平的差异,进一步验证这两对基因启动子的突变对相应基因转录水平的影响。【结果】Quantitative Real Time PCR检测结果显示H37RapabB启动子活性是H37Rv pabB启动子活性的6倍(p<0.05),而H37Rv lpdA启动子的活性是H37RalpdA启动子的2倍(p<0.05)。【结论】pabB,lpdA的启动子在H37Ra中的突变对其启动子的活性产生了影响,其中lpdA启动子的突变可能与结核分枝杆菌H37Ra的毒力丧失有关。
【Objective】 By analyzing the whole genome sequence of non-virulent strain of Mycobacterium tuberculosis H37Ra and comparing with the H37Rv genome sequence, it was found that the predicted promoter regions of pabB and lpdA were mutated. We use the reporter gene to confirm the relationship between the promoter mutation and its gene transcription and to explore the underlying causes of the virulence of Mycobacterium tuberculosis H37Ra. 【Method】 The promoter regions of these two pairs of genes were predicted by using bioinformatics methods. The promoters of these two pairs of genes were cloned by PCR and ligated with the promoter of mycobacterial promoter pMC210. After DNA sequencing confirmed that the fragments were correct, The recombinant plasmid was transformed into M. smegmatis mc2155 by electroporation. Quantitative Real-Time RT-PCR detection lacZ reporter gene transcription level differences further verify the two pairs of gene promoter mutations on the corresponding gene transcription level. 【Results】 The results of Quantitative Real Time PCR showed that the H37RababB promoter activity was 6 times higher than that of the H37Rv pabB promoter (p <0.05), while the H37Rv lpdA promoter activity was twice as high as that of the H37RalpdA promoter (p <0.05). 【Conclusion】 The mutation of pabB and lpdA promoter in H37Ra has an impact on its promoter activity. The mutation of lpdA promoter may be related to the loss of virulence of M. tuberculosis H37Ra.