结核分枝杆菌无毒株H37Ra启动子突变基因的比较分析

发布时间：2018-08-26 09:41

【摘要】： 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,发现一些基因的启动子区域发生了突变,我们利用分枝杆菌启动子探针载体pMC210,分别构建了结核分枝杆菌H37Ra和H37Rv的六个重要基因(sec、pabB、phoH2、sigC、nrdH、lpdA)的启动子探针重组载体。利用报告基因lacZ检测突变前后启动子的活性变化。同时,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六对基因(sec、pabB、phoH2、sigC、nrdH、lpdA)的启动子区域,采用PCR技术克隆这六对基因的启动子,双酶切后与分枝杆菌启动子探针载体pMC210对应的双酶切片段相连,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中。通过体外测定β-半乳糖苷酶的活性来评估启动子的强度和Quantitative Real-Time RT-PCR的方法检测报告基因lacZ的转录水平差异,检测启动子的突变对相应基因转录水平的影响。 结果通过PCR和构建克隆测序比对发现基因sec、phoH2、sigC、nrdH的启动子与GenBank上提交的序列存在差异,结核分枝杆菌无毒株H37Ra和有毒株H37Rv相比较,基因sec、phoH2、sigC、nrdH预测的启动子并没有突变。只有基因pabB和lpdA存在启动子突变。H37Rv和H37Ra的pabB基因的启动子探针重组载体转化耻垢分枝杆菌后,体外测定的β-半乳糖苷酶的活性较弱,无统计学意义,而Quantitative Real Time PCR检测结果显示H37Ra pabB启动子调节报告基因lacZ转录的活性是H37Rv pabB启动子的6倍(p0.05),H37Rv和H37Ra lpdA启动子探针重组载体体外测得的β-半乳糖苷酶的活性和Real time PCR结果均显示H37Rv lpdA启动子调节报告基因lacZ表达的活性比H37Ra lpdA启动子高2倍(p0.05)左右。 结论pabB,lpdA的启动子在H37Ra中的突变对其启动子活性的产生了显著的影响,并且lpdA启动子突变可能与结核分枝杆菌H37Ra的毒力丧失有关。
[Abstract]:Objective by analyzing the whole genome sequence of Mycobacterium tuberculosis H37Ra, and comparing it with the H37Rv genome sequence, we found that the promoter region of some genes was mutated. The recombinant vectors of six important genes (sec,pabB,phoH2,sigC,nrdH,lpdA) of Mycobacterium tuberculosis (H37Ra) and H37Rv (sec,pabB,phoH2,sigC,nrdH,lpdA) were constructed by using Mycobacterium tuberculosis promoter probe vector pMC210,. The promoter activity before and after mutation was detected by reporter gene lacZ. At the same time, the relationship between promoter mutation and transcriptional level of its gene was confirmed, and the internal cause of H37Ra virulence loss in Mycobacterium tuberculosis was explored. Methods the promoter regions of the six pairs of genes (sec,pabB,phoH2,sigC,nrdH,lpdA) were predicted by bioinformatics. The promoters of the six pairs of genes (sec,pabB,phoH2,sigC,nrdH,lpdA) were cloned by PCR technique. The recombinant plasmid was transformed into mc2155 by electroporation after double enzyme digestion and sequencing of the double enzyme fragment corresponding to the promoter probe vector pMC210 of Mycobacterium spp. The activity of 尾 -galactosidase was measured in vitro to evaluate the intensity of promoter and the method of Quantitative Real-Time RT-PCR to detect the difference of transcription level of reporter gene lacZ, and to detect the effect of promoter mutation on the transcription level of the corresponding gene. Results the promoter of gene sec,phoH2,sigC,nrdH was different from the sequence submitted on GenBank by PCR and construction of clone sequencing. The promoter predicted by gene sec,phoH2,sigC,nrdH had no mutation compared with that of H37Rv from Mycobacterium tuberculosis non-virulent strain (H37Ra) and virulent strain (H37Rv). Only pabB and lpdA had promoter mutation. H37Rv and H37Ra pabB gene promoter probe recombinant vector transformed Mycobacterium smearus, the activity of 尾 -galactosidase was weak in vitro, and had no statistical significance. The results of Quantitative Real Time PCR analysis showed that the activity of H37Ra pabB promoter regulating the transcription of reporter gene lacZ was 6 times of that of H37Rv pabB promoter and the activity of 尾 -galactosidase detected by H37Ra lpdA promoter probe and 尾 -galactosidase in vitro and Real time PCR result. The results showed that the activity of H37Rv lpdA promoter regulating reporter gene lacZ was about 2 times higher than that of H37Ra lpdA promoter (p0. 05). Conclusion the promoter mutation of pabB,lpdA in H37Ra has a significant effect on its promoter activity, and the lpdA promoter mutation may be related to the loss of virulence of Mycobacterium tuberculosis H37Ra.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378.911

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