双组份信号转导系统反应调节子DevR的表达与结核分枝杆菌耐药的关系研究

发布时间：2018-03-30 11:11

  本文选题：结核分枝杆菌　切入点：结核病　出处：《第四军医大学》2015年博士论文

【摘要】：背景结核病是一种严重危害公众健康和威胁人类生存的传染病。我国是全球结核病疫情最严重的国家之一,也是结核病耐药负担最重的国家之一。因此研究结核病的耐药机制,对于结核病的防控具有重要意义。我们前期利用LC-MS/MS对临床分离的结核分枝杆菌敏感株、异烟肼单耐药菌株和耐多药菌株的差异表达蛋白进行检测,结果发现结核分枝杆菌双组份信号转导系统的反应调节子Dev R在临床分离的耐多药菌株中表达明显升高。Dev R是结核分枝杆菌重要的休眠调控因子,而休眠能导致结核菌对多种药物耐药,提示Dev R可能与结核分枝杆菌耐药密切相关。因此,本课题拟在前期工作的基础之上,进一步验证Dev R的表达与结核分枝杆菌耐药的关系。目的1.鉴定结核分枝杆菌dev R的表达水平与药敏表型的关系。2.明确不同耐药表型菌株的遗传背景对dev R的表达影响。3.阐明dev R的表达变化对结核分枝杆菌耐药性的影响。方法1.将培养至对数生长早期的32株结核分枝杆菌敏感菌株、18株异烟肼单耐药菌株、2株利福平单耐药菌株和45株耐多药菌株的总RNA进行提取,然后利用荧光定量PCR检测dev R的表达水平;分别向培养至对数长早期的6株敏感菌株中,分别加入其1/2 MIC浓度的异烟肼、利福平、链霉素或乙胺丁醇,以7H9培养基作为对照,诱导培养2小时后,检测dev R基因的表达量。2.对97株临床分离的结核分枝杆菌菌株及标准株H37Rv株进行基因组DNA的提取;然后,对菌株基因组DNA的15个MIRU-VNTR位点进行PCR扩增,并将扩增结果上传至MIRU-VNTR Plus数据库;最终,利用系统基因树分析对菌株的基因型进行鉴定。3.利用Devsilab rep-PCR对97株结核分枝杆菌菌株的基因组DNA进行扩增,以获得每株菌的DNA指纹图谱;最终,利用Deversilab在线分析软件(Version 3.4)将所有菌株DNA指纹数据进行聚类分析。4.对dev R上游的启动子区进行预测,并利用PCR对97株临床分离菌株的dev R启动子区进行扩增;然后将扩增产物进行测序鉴定并进行比对分析。5.将可表达噬菌体重组酶Che C 60-61的质粒p JV53电转化入M.bovis BCG菌株,以获得重组的M.bovis BCG菌株。6.将dev R的上、下游同源臂连接在博莱霉素筛选标签的两侧,以获得dev R的突变子;然后,将dev R的突变子电转化入重组的M.bovis BCG菌株;最后,利用PCR和测序鉴定dev R敲除菌株。7.利用结核分枝杆菌穿梭表达载体p MN437构建dev R的过表达载体;然后,将dev R过表达载体Pmn437dev R电转化入M.bovis BCG菌株;最终,利用PCR和荧光显微镜等技术对dev R的过表达菌株进行筛选和鉴定。8.利用刃天青显色药敏法检测野生型BCG、dev R高表达菌株和dev R缺失表达菌株的MIC。结果1.定量PCR的检测结果显示:在INH单耐药菌株和MDR菌株中,dev R的表达量明显升高(p0.05),分别是敏感株的1.42倍和7.36倍。INH、RIF和EMB菌株分别诱导临床分离的敏感菌株后,dev R的表达量明显升高,其平均相对表达量均超过了对照组的2倍。2.MIRU-VNTR基因分型结果显示:本研究所收集的临床分离菌株均为北京基因型,但本实验所保存的标准菌株H37Rv株与H37Rv原始株比较,在MIRU-26、Mtub39和QUB4156位点上出现了重复序列拷贝数的变异,但经系统基因树鉴定仍属于H37Rv菌株。3.将Devsilab rep-PCR获得的DNA指纹图谱聚类分析,结果显示:本实验室所保存的结核分枝杆菌菌株的基因型呈现明显的多态性;每个样本具有独特DNA指纹图谱,所有菌株的总体的相似度为55%;以70%为cut-off值可将所有菌株分为8个主要的基因型,但进一步的分析发现菌株基因型与其耐药表型之间的相关性不具有统计学意义(p0.05);此外,对临床分离菌株的基因型(rep-PCR)与dev R表达量的相关性分析显示:dev R在耐药菌株和敏感菌株间的差异表达与基因型相的相似度无关(p0.05)。4.对dev R上游792bp的基因片段的测序,结果显示:有22株菌(6敏感、4异烟肼单耐药、12株耐多药菌株)发生了一个A碱基的缺失突变;该突变位于dev R上游-370bp处,且属首次发现。此外,dev R的表达与基因突变的相关性关系分析提示:dev R在突变株和野生株之间不存在表达差异(p0.05)。5.本研究利用同源重组技术成功构建了dev R缺失表达的M.bovis BCG菌株;本研究也成功构建了dev R过表达的M.bovis BCG菌株。6.野生型M.bovis BCG株、dev R缺失株和dev R过表达株的MIC检测结果显示:野生株分别对异烟肼、利福平、链霉素和乙胺丁醇的MIC为0.06、0.17、0.5和2.6μg/ml;dev R缺失株分别对异烟肼、利福平、链霉素和乙胺丁醇的MIC为0.06、0.20、0.33和3.3μg/ml;dev R过表达株分别对INH、RIF、SM和EMB的MIC为0.08、0.20、0.33和3.3μg/ml。即,这三种菌株均对一线抗结核药物敏感。结论本课题系统地分析了dev R基因在多种耐药表型菌株中的表达情况,首次发现并证实了dev R在结核分枝杆菌耐药菌株中表达明显升高,且INH、SM和EMB均可诱导其表达。并揭示了dev R在耐药菌株和敏感菌之间的表达差异不受菌株基因型的影响,提示其表达变化与耐药密切相关。本课题利用同源重组技术成功得对M.bovis BCG菌株进行了dev R基因敲除,并构建了dev R的过表达菌株,可以为下一步研究dev R的表达变化与耐药性突变的关系奠定了坚实地工作基础。
[Abstract]:Background: tuberculosis is a serious public health hazard and threat to the survival of the human infectious disease. China is one of the most serious global TB epidemic in the country, and it is also one of the countries with the highest burden of TB drug resistance. Therefore study on resistance mechanism of tuberculosis, the tuberculosis prevention and control has important significance. Our previous use of LC-MS/MS of Mycobacterium tuberculosis in clinical isolated strains, single resistant strains of isoniazid and multi drug resistant strains of differences in protein expression were detected, the results found that Mycobacterium tuberculosis two-component signal transduction system response regulator Dev R increased.Dev R of Mycobacterium tuberculosis is an important factor in the regulation of expression of dormant clinical isolated multi drug resistant strains, and sleep can lead to Mycobacterium tuberculosis resistant to multiple drugs, suggesting that Dev R may be closely related to Mycobacterium tuberculosis drug resistance. Therefore, this paper do the preparatory work for the group Based on the relationship between Dev R expression and further resistant Mycobacterium tuberculosis test. Expression of expression of genetic background of the relationship between.2. expression level to 1. identification of Mycobacterium tuberculosis dev R and drug susceptibility phenotype of different resistant phenotypes of dev strain R.3. dev R to clarify the effect of drug resistance of Mycobacterium tuberculosis. Methods 1. cultured to the logarithmic growth of the early 32 Mycobacterium tuberculosis sensitive strains, 18 isoniazid resistant strains, 2 strains of RNA resistant strains and 45 strains of rifampin single multi drug resistant strains were extracted, and then use the detection of the expression levels of dev R fluorescence quantitative PCR; were cultured to logarithmic long early 6 strain sensitive strains, were added to the 1/2 MIC of isoniazid, rifampicin concentration, streptomycin and ethambutol, with 7H9 medium as the control, after 2 hours of culturing, the detection of dev expression of R gene of 97 strains of.2. The extraction of clinical isolates of Mycobacterium tuberculosis strains and H37Rv strains of genomic DNA; then, the 15 MIRU-VNTR loci of genomic DNA was amplified by PCR, and the amplified results uploaded to MIRU-VNTR Plus database; finally, analysis of genotype.3. strains were identified by Devsilab rep-PCR genomic DNA of 97 strains of Mycobacterium tuberculosis Mycobacterium strains were amplified using gene tree, in order to obtain the DNA fingerprint of each bacteria; finally, using Deversilab software online (Version 3.4) all strain DNA fingerprint data clustering analysis of.4. promoter region of dev upstream of the R prediction, and the use of PCR against 97 clinical isolates of dev the R promoter region was amplified; then the amplified products were sequenced and analyzed.5. expression of bacteriophage recombinase Che C 60-61 plasmid P JV53 was transformed into M.bovi S BCG strain to obtain recombinant M.bovis BCG strain.6. dev R on the downstream homologous arm connects the two screening tag in bleomycin, to obtain dev mutant R; then, the M.bovis BCG dev mutant R strains will power into the reorganization; finally, using PCR and sequencing dev R knock in addition to the use of.7. strains of Mycobacterium tuberculosis shuttle expression vector p to construct expression vector of MN437 dev R; then, dev R expression vector Pmn437dev R was transformed into M.bovis BCG strain; finally, expression and identification of.8. strains were screened using the resazurin chromogenic drug sensitivity assay of wild type BCG on dev by R PCR and fluorescence microscopy, R strain and dev strain MIC. with high dev expression loss of R expression results of the testing results of 1. quantitative PCR shows that in INH resistant strains and MDR strains, the expression of dev R increased significantly (P0.05), respectively is 1.4 sensitive strains. 2 times and 7.36 times of.INH, RIF and EMB strains were sensitive strains of clinical isolates after induction, expression of dev R increased significantly, the average relative expression of.2.MIRU-VNTR gene were 2 times more than the control group the genotyping results showed that: the clinical isolates collected in the present study are Beijing genotype, but save the experimental standard strain H37Rv H37Rv strain and the original strain in comparison, MIRU-26, Mtub39 and QUB4156 loci appeared on the copy number of repeat sequence variation, but the system still belongs to the gene tree identification H37Rv strain.3. analysis, clustering DNA fingerprint Devsilab rep-PCR results showed that genotypes preserved in our laboratory tuberculosis Mycobacterium strains showed obvious polymorphism; each sample has a unique DNA fingerprint, the overall similarity of all strains was 55%; with 70% cut-off value of all strains were divided into 8 major genes Type, but further analysis found that the correlation between genotype and drug resistance phenotype was not statistically significant (P0.05); in addition, the genotypes of clinical isolates (rep-PCR) and dev R expression of R showed that dev in drug-resistant strains and expression difference between sensitive strain and genotype phase independent similarity (P0.05) sequencing,.4. to Dev upstream of the R 792bp gene showed that 22 strains (6 4 INH sensitive, single drug resistance, 12 strains of multi drug resistant strains) had a A deletion mutation; the mutation at dev upstream of R -370bp, and is the first time. In addition, the correlation analysis of the relationship between dev expression and gene mutation of R in R suggest that dev expression differences between mutant and wild-type (P0.05).5. using homologous recombination technique to construct M.bovis BCG strain expressing dev R deletion; this study also successfully R M.bovis BCG constructed dev strain.6. strain expressing wild type M.bovis BCG, dev R and dev R deletion mutant MIC overexpression strain test results show: wild strains of isoniazid, rifampicin, streptomycin and ethambutol MIC 0.06,0.17,0.5 and 2.6 g/ml respectively; dev R mutant of isoniazid, rifampicin streptomycin and ethambutol, MIC 0.06,0.20,0.33 and 3.3 g/ml; dev R strains of INH, the expression of RIF, SM and EMB MIC 0.08,0.20,0.33 and 3.3 g/ml., the three strains were sensitive to first-line anti tuberculosis drugs. Conclusion this paper systematically analyzed the expression of dev R gene in various the resistance phenotype in strains, first discovered and confirmed the expression of dev R in drug-resistant strains of Mycobacterium tuberculosis were significantly increased, and INH, SM and EMB can induce the expression of dev R. And revealed the expression between resistant strains and the sensitive strain difference from strain The influence of genotype, suggesting that its expression and resistance are closely related. The project successfully to M.bovis BCG strain dev R gene knockout by homologous recombination technology, and construct the over expression strain dev R, the relationship can be mutated to expression of drug resistance and the next step for the research of dev R has laid a solid foundation to work.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R446.5
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本文编号：1685664