本文選題：結核分枝桿菌　切入點：結核病　出處：《第四軍醫(yī)大學》2015年博士論文

【摘要】：背景結核病是一種嚴重危害公眾健康和威脅人類生存的傳染病。我國是全球結核病疫情最嚴重的國家之一,也是結核病耐藥負擔最重的國家之一。因此研究結核病的耐藥機制,對于結核病的防控具有重要意義。我們前期利用LC-MS/MS對臨床分離的結核分枝桿菌敏感株、異煙肼單耐藥菌株和耐多藥菌株的差異表達蛋白進行檢測,結果發(fā)現(xiàn)結核分枝桿菌雙組份信號轉導系統(tǒng)的反應調節(jié)子Dev R在臨床分離的耐多藥菌株中表達明顯升高。Dev R是結核分枝桿菌重要的休眠調控因子,而休眠能導致結核菌對多種藥物耐藥,提示Dev R可能與結核分枝桿菌耐藥密切相關。因此,本課題擬在前期工作的基礎之上,進一步驗證Dev R的表達與結核分枝桿菌耐藥的關系。目的1.鑒定結核分枝桿菌dev R的表達水平與藥敏表型的關系。2.明確不同耐藥表型菌株的遺傳背景對dev R的表達影響。3.闡明dev R的表達變化對結核分枝桿菌耐藥性的影響。方法1.將培養(yǎng)至對數生長早期的32株結核分枝桿菌敏感菌株、18株異煙肼單耐藥菌株、2株利福平單耐藥菌株和45株耐多藥菌株的總RNA進行提取,然后利用熒光定量PCR檢測dev R的表達水平;分別向培養(yǎng)至對數長早期的6株敏感菌株中,分別加入其1/2 MIC濃度的異煙肼、利福平、鏈霉素或乙胺丁醇,以7H9培養(yǎng)基作為對照,誘導培養(yǎng)2小時后,檢測dev R基因的表達量。2.對97株臨床分離的結核分枝桿菌菌株及標準株H37Rv株進行基因組DNA的提取;然后,對菌株基因組DNA的15個MIRU-VNTR位點進行PCR擴增,并將擴增結果上傳至MIRU-VNTR Plus數據庫;最終,利用系統(tǒng)基因樹分析對菌株的基因型進行鑒定。3.利用Devsilab rep-PCR對97株結核分枝桿菌菌株的基因組DNA進行擴增,以獲得每株菌的DNA指紋圖譜;最終,利用Deversilab在線分析軟件(Version 3.4)將所有菌株DNA指紋數據進行聚類分析。4.對dev R上游的啟動子區(qū)進行預測,并利用PCR對97株臨床分離菌株的dev R啟動子區(qū)進行擴增;然后將擴增產物進行測序鑒定并進行比對分析。5.將可表達噬菌體重組酶Che C 60-61的質粒p JV53電轉化入M.bovis BCG菌株,以獲得重組的M.bovis BCG菌株。6.將dev R的上、下游同源臂連接在博萊霉素篩選標簽的兩側,以獲得dev R的突變子;然后,將dev R的突變子電轉化入重組的M.bovis BCG菌株;最后,利用PCR和測序鑒定dev R敲除菌株。7.利用結核分枝桿菌穿梭表達載體p MN437構建dev R的過表達載體;然后,將dev R過表達載體Pmn437dev R電轉化入M.bovis BCG菌株;最終,利用PCR和熒光顯微鏡等技術對dev R的過表達菌株進行篩選和鑒定。8.利用刃天青顯色藥敏法檢測野生型BCG、dev R高表達菌株和dev R缺失表達菌株的MIC。結果1.定量PCR的檢測結果顯示:在INH單耐藥菌株和MDR菌株中,dev R的表達量明顯升高(p0.05),分別是敏感株的1.42倍和7.36倍。INH、RIF和EMB菌株分別誘導臨床分離的敏感菌株后,dev R的表達量明顯升高,其平均相對表達量均超過了對照組的2倍。2.MIRU-VNTR基因分型結果顯示:本研究所收集的臨床分離菌株均為北京基因型,但本實驗所保存的標準菌株H37Rv株與H37Rv原始株比較,在MIRU-26、Mtub39和QUB4156位點上出現(xiàn)了重復序列拷貝數的變異,但經系統(tǒng)基因樹鑒定仍屬于H37Rv菌株。3.將Devsilab rep-PCR獲得的DNA指紋圖譜聚類分析,結果顯示:本實驗室所保存的結核分枝桿菌菌株的基因型呈現(xiàn)明顯的多態(tài)性;每個樣本具有獨特DNA指紋圖譜,所有菌株的總體的相似度為55%;以70%為cut-off值可將所有菌株分為8個主要的基因型,但進一步的分析發(fā)現(xiàn)菌株基因型與其耐藥表型之間的相關性不具有統(tǒng)計學意義(p0.05);此外,對臨床分離菌株的基因型(rep-PCR)與dev R表達量的相關性分析顯示:dev R在耐藥菌株和敏感菌株間的差異表達與基因型相的相似度無關(p0.05)。4.對dev R上游792bp的基因片段的測序,結果顯示:有22株菌(6敏感、4異煙肼單耐藥、12株耐多藥菌株)發(fā)生了一個A堿基的缺失突變;該突變位于dev R上游-370bp處,且屬首次發(fā)現(xiàn)。此外,dev R的表達與基因突變的相關性關系分析提示:dev R在突變株和野生株之間不存在表達差異(p0.05)。5.本研究利用同源重組技術成功構建了dev R缺失表達的M.bovis BCG菌株;本研究也成功構建了dev R過表達的M.bovis BCG菌株。6.野生型M.bovis BCG株、dev R缺失株和dev R過表達株的MIC檢測結果顯示:野生株分別對異煙肼、利福平、鏈霉素和乙胺丁醇的MIC為0.06、0.17、0.5和2.6μg/ml;dev R缺失株分別對異煙肼、利福平、鏈霉素和乙胺丁醇的MIC為0.06、0.20、0.33和3.3μg/ml;dev R過表達株分別對INH、RIF、SM和EMB的MIC為0.08、0.20、0.33和3.3μg/ml。即,這三種菌株均對一線抗結核藥物敏感。結論本課題系統(tǒng)地分析了dev R基因在多種耐藥表型菌株中的表達情況,首次發(fā)現(xiàn)并證實了dev R在結核分枝桿菌耐藥菌株中表達明顯升高,且INH、SM和EMB均可誘導其表達。并揭示了dev R在耐藥菌株和敏感菌之間的表達差異不受菌株基因型的影響,提示其表達變化與耐藥密切相關。本課題利用同源重組技術成功得對M.bovis BCG菌株進行了dev R基因敲除,并構建了dev R的過表達菌株,可以為下一步研究dev R的表達變化與耐藥性突變的關系奠定了堅實地工作基礎。
[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.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R446.5
，

本文編號：1685664