【摘要】： 目的通過分析結(jié)核分枝桿菌無毒株H37Ra的全基因組序列,并與H37Rv基因組序列比較,發(fā)現(xiàn)一些基因的啟動子區(qū)域發(fā)生了突變,我們利用分枝桿菌啟動子探針載體pMC210,分別構(gòu)建了結(jié)核分枝桿菌H37Ra和H37Rv的六個重要基因(sec、pabB、phoH2、sigC、nrdH、lpdA)的啟動子探針重組載體。利用報告基因lacZ檢測突變前后啟動子的活性變化。同時,確認啟動子突變與其基因轉(zhuǎn)錄水平的關(guān)系,探索結(jié)核分枝桿菌H37Ra毒力喪失的內(nèi)在原因。 方法利用生物信息學方法預(yù)測這六對基因(sec、pabB、phoH2、sigC、nrdH、lpdA)的啟動子區(qū)域,采用PCR技術(shù)克隆這六對基因的啟動子,雙酶切后與分枝桿菌啟動子探針載體pMC210對應(yīng)的雙酶切片段相連,DNA測序證實連接片段正確后,用電穿孔法將重組質(zhì)粒轉(zhuǎn)化至恥垢分枝桿菌mc2155中。通過體外測定β-半乳糖苷酶的活性來評估啟動子的強度和Quantitative Real-Time RT-PCR的方法檢測報告基因lacZ的轉(zhuǎn)錄水平差異,檢測啟動子的突變對相應(yīng)基因轉(zhuǎn)錄水平的影響。 結(jié)果通過PCR和構(gòu)建克隆測序比對發(fā)現(xiàn)基因sec、phoH2、sigC、nrdH的啟動子與GenBank上提交的序列存在差異,結(jié)核分枝桿菌無毒株H37Ra和有毒株H37Rv相比較,基因sec、phoH2、sigC、nrdH預(yù)測的啟動子并沒有突變。只有基因pabB和lpdA存在啟動子突變。H37Rv和H37Ra的pabB基因的啟動子探針重組載體轉(zhuǎn)化恥垢分枝桿菌后,體外測定的β-半乳糖苷酶的活性較弱,無統(tǒng)計學意義,而Quantitative Real Time PCR檢測結(jié)果顯示H37Ra pabB啟動子調(diào)節(jié)報告基因lacZ轉(zhuǎn)錄的活性是H37Rv pabB啟動子的6倍(p0.05),H37Rv和H37Ra lpdA啟動子探針重組載體體外測得的β-半乳糖苷酶的活性和Real time PCR結(jié)果均顯示H37Rv lpdA啟動子調(diào)節(jié)報告基因lacZ表達的活性比H37Ra lpdA啟動子高2倍(p0.05)左右。 結(jié)論pabB,lpdA的啟動子在H37Ra中的突變對其啟動子活性的產(chǎn)生了顯著的影響,并且lpdA啟動子突變可能與結(jié)核分枝桿菌H37Ra的毒力喪失有關(guān)。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R378.911

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