【作者】 孙青；

【导师】 赵英伟；

【作者基本信息】 苏州大学 ， 病原生物学， 2010， 硕士

【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(p<0.05),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(p<0.05)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。更多还原

【Abstract】 Objective Comparative genomic study revealed mutations at the promoter regions of six genes (sec, pabB, phoH2, sigC, nrdH, lpdA) in avirulent Mycobacterium tuberculosis strain H37Ra compared to its virulent counterpart strain H37Rv. This study used mycobacterial promoter probe vector pMC210 to construct six genes’promoter probe vectors of strains H37Ra and H37Rv. This study used lacZ reporter gene to test whether those mutations will affect promoter activity and its potential relationship to virulence attenuation of H37Ra.Methods Promoter sequences of six genes were predicted by the“Neural Network Promoter Prediction”method (http://www.fruitfly.org/seq_tools/promoter.html). Promoter regions were amplified and inserted into mycobacterial promoter probe vector pMC210. Resultant recombinant plasmids were transformed into M. smegmatis by electroporation. This study assessed the strength of the promoter through determination ofβ-galactosidase activity. The transcription activity of lacZ in the control of cloned promoters was monitored by Quantitative Real-Time RT-PCR.Results The promoter sequences of sec, phoH2, sigC, nrdH which were submitted in the GenBank were probably controversial. Quantitative Real Time PCR or determination ofβ-galactosidase activity results showed that the promoter activity of H37Ra pabB was six times more than that of H37Rv pabB (p < 0.05), while the promoter activity of the H37Rv lpdA was two fold of that of H37Ra lpdA (p < 0.05).Conclusion The mutations in pabB and lpdA promoters affect its expression activity significantly. The T-A mutation in lpdA promoter may be related to virulence attenuation of H37Ra.更多还原