發(fā)布時間：2018-01-25 05:58

  本文關(guān)鍵詞： 結(jié)核分枝桿菌 mazEF 毒素-抗毒素系統(tǒng) 過表達 潛伏感染 實時定量PCR技術(shù) 診斷　出處：《石河子大學》2016年碩士論文　論文類型：學位論文

【摘要】：目的:(1).構(gòu)建北京、非北京基因型結(jié)核分枝桿菌、標準株(H37RV)的mazEF3、mazEF6、mazEF9基因的過表達菌株,觀測其在低氧、營養(yǎng)缺乏條件下的生存能力,同時檢測不同電轉(zhuǎn)電壓下過表達菌株相應基因的mRNA表達水平。(2).檢測預測的7種細胞因子的mRNA的表達水平,探討目的因子分子表達水平作為結(jié)核分枝桿菌潛伏感染診斷的生物標志物的可能性。方法:(1).利用大腸桿菌-結(jié)核分枝桿菌穿梭表達質(zhì)粒pMV361,構(gòu)建出重組質(zhì)粒pMV361-mazEF3、pMV361-mazEF6、pMV361-mazEF9;電穿孔將重組質(zhì)粒導入北京基因型、非北京基因型結(jié)核分枝桿菌中。在卡那霉素培養(yǎng)基上初步篩選,提取陽性菌株的質(zhì)粒進行PCR、雙酶切,送公司測序,驗證構(gòu)建是否成功;檢測不同菌株在不同生長條件下的生長情況。實時熒光定量PCR技術(shù)檢測不同電轉(zhuǎn)電壓下mazEF3的mRNA表達水平,尋找構(gòu)建mazEF3過表達菌株的最佳電轉(zhuǎn)電壓。(2).健康對照組64人,活動性肺結(jié)核50人,結(jié)核分枝桿菌潛伏感染60人,實時熒光定量PCR(qRT-PCR)檢測結(jié)核特異性抗原刺激的外周血中TNF-α、IFN-γ、IL-2、IL-10、IFI35、IP10及Foxp3的mRNA表達水平。結(jié)果:(1).成功構(gòu)建了H37RV、北京、非北京基因型的結(jié)核分枝桿菌的mazEF3、mazEF6、mazEF9過表達菌株。北京基因型的結(jié)核分枝桿菌在正常和應激環(huán)境下,生長狀態(tài)均好于非北京基因型結(jié)核分枝桿菌。實時熒光定量檢測,電轉(zhuǎn)電壓為2300V時,構(gòu)建的過表達菌株mazEF3的mRNA表達量最高。(2).7種因子相對表達量在活動性肺結(jié)核組為2.33±0.09、4.94±0.45、150±17.82、2.02±0.55、9.53±5.96、5.56±0.97、1.24±0.17,潛伏感染組分別為3.2±0.61、10.04±1.89、202±17.6、21.89±10.25、29.17±11.19、41.35±11.46、2.14±0.67,健康組分別為1.2±0.08、2.92±0.03、53±17.82、1.48±0.17、1.28±0.77、1.63±0.25、1.03±0.67。潛伏感染組與健康組相比7種細胞因子的mRNA表達水平,差異均有統(tǒng)計學意義(P0.01),相應細胞因子檢測的敏感性、特異性分別為95.1%、82.5%,92.5%、88.7%,91.9%、90.1%,88.2%、51.5%,74.2%、61.2%,81.8%、73.4%和80.4%、51.3%;潛伏感染組和活動性結(jié)核組比較除IFI35因子外,其余6種細胞因子的mRNA表達水平差異均有統(tǒng)計學意義(P0.05),相應細胞因子檢測的特異性、敏感性分別為90.6%、80%,88.6%、92.7%,91.9%、86.2%,57.6%、60%,50%、58%,76%、72.2%和66.7%、67.1%;健康對照組和活動性結(jié)核組相比較,7種細胞因子的mRNA表達水平差異均有統(tǒng)計學意義(P0.01),相應細胞因子檢測的敏感性、特異性分別為92.3%、90%,88.6%、86.7%,94.4%、86.7%,89.7%、65.8%,82.4%、63.3%,88.2%、70%和92.9%、72.1%。結(jié)核病密切接觸者潛伏感染率為48.97%,健康組潛伏感染率為36%。結(jié)論:(1).成功構(gòu)建了北京、非北京基因型的mazEF3、mazEF6、mazEF9過表達菌株,2300V是mazEF3的最佳電轉(zhuǎn)電壓。(2).結(jié)核病患者血清TNF-α、IFN-γ、IL-2 mRNA水平升高,以潛伏感染者升高更顯著,因此可作為診斷結(jié)核分枝桿菌潛伏感染的生物標志物。
[Abstract]:Objective to construct the overexpression strain of mazEF3, mazEF6, mazEF9 gene from Mycobacterium tuberculosis, a non-Beijing genotype of Mycobacterium tuberculosis, standard strain H37RV. Its viability was observed under the condition of hypoxia and lack of nutrition. At the same time, the mRNA expression level of the corresponding gene was detected under different electroporation voltages, and the mRNA expression level of 7 cytokines was detected. Objective to explore the possibility of using the expression level of factors as a biomarker for the diagnosis of latent infection of Mycobacterium tuberculosis. Methods the expression plasmid pMV361 of Escherichia coli and Mycobacterium tuberculosis shuttle was used. A recombinant plasmid pMV361-mazEF3 pMV361-mazEF6 pMV361-mazEF9 was constructed. The recombinant plasmid was introduced into Mycobacterium tuberculosis of Beijing genotype and non-Beijing genotype by electroporation. The plasmids of positive strains were extracted from kanamycin medium for PCR.The plasmid was digested by double enzyme and sent to the company for sequencing. Verify that the build is successful; To detect the growth of different strains under different growth conditions, real-time fluorescence quantitative PCR technique was used to detect the mRNA expression level of mazEF3 under different voltages. Objective: to find out the best electroporation voltage of mazEF3 overexpression strain. 64 healthy control group, 50 active pulmonary tuberculosis and 60 mycobacterium tuberculosis latent infection. Real-time quantitative PCR- QRT-PCR was used to detect TNF- 偽 IFN- 緯 IL-2IL-10 IL-10IFI35 in peripheral blood stimulated by TB specific antigen. The mRNA expression level of IP10 and Foxp3. Results the H37RV, Beijing, non-Beijing genotype of Mycobacterium tuberculosis mazEF3 and mazEF6 were successfully constructed. MazEF9 overexpression strains. Mycobacterium tuberculosis of Beijing genotype in normal and stress environment, the growth state of Mycobacterium tuberculosis is better than that of non-Beijing genotype Mycobacterium tuberculosis. Real time fluorescence quantitative detection. When the voltage of electroporation was 2300V, the relative expression of mRNA was 2.33 鹵0.09 in active pulmonary tuberculosis group. 4.94 鹵0.45U 150 鹵17.82U 2.02 鹵0.55U 9.53 鹵5.96U 5.56 鹵0.97U 1.24 鹵0.17. The latent infection group was 3.2 鹵0.61g 10.04 鹵1.89 鹵1.89 鹵21.89 鹵10.25 鹵29.17 鹵11.19, respectively. 41.35 鹵11.46 鹵2.14 鹵0.67 in the control group and 1.2 鹵0.08 鹵2.92 鹵0.03 鹵53 鹵17.82 鹵1.48 鹵0.17 in the healthy group, respectively. 1.28 鹵0.77 ~ 1.63 鹵0.25 ~ 1.03 鹵0.67. The mRNA expression level of 7 cytokines in latent infection group was higher than that in healthy group. The difference was statistically significant (P 0.01). The sensitivity and specificity of the corresponding cytokines were 92.5% and 92.5%, respectively. The sensitivity of the corresponding cytokines was 91.9%. There were 71.2% and 83.2% and 83.4% and 80.4%, 51.3% and 81.4%, 51.5% and 74.2%, 83.2% and 83.4%, 51.3% and 80.4%, 51.3% and 80.4%, respectively. There were significant differences in the expression of mRNA between latent infection group and active tuberculosis group except IFI35 factor (P 0.05). The specificity and sensitivity of the corresponding cytokine detection were 90.6 and 88.6and 91.9% and 91.9%, respectively. The sensitivity of the corresponding cytokines was 57.6%. There were 72.2% and 66.7%, 67.1% and 67.7%, respectively. The mRNA expression level of 7 cytokines in healthy control group and active tuberculosis group was significantly higher than that in active tuberculosis group (P 0.01). The specificity was 92.3% and 88.6% respectively, including 86.7% and 86.7%, and 86.7% and 85.7%, 85.3% and 83.2%, respectively. The latent infection rate of close contact with tuberculosis was 48.97 and that of healthy group was 360.Conclusion Beijing was successfully constructed. The overexpression strain of mazEF3 / mazEF6 / mazEF9, which is not a Beijing genotype, is the best electroporation voltage of mazEF3. The serum TNF- 偽 of patients with tuberculosis is TNF- 偽. The level of IL-2 mRNA in IFN- 緯 is higher than that in latent infection, so it can be used as a biomarker for the diagnosis of latent infection of Mycobacterium tuberculosis.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R378.911

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1 屈艷琳;結(jié)核分枝桿菌mazEF基因及結(jié)核病生物標志物的探究[D];石河子大學;2016年

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本文編號：1462185