结核分枝杆菌mazEF基因及结核病生物标志物的探究

发布时间：2018-01-25 05:58

本文关键词： 结核分枝杆菌 mazEF 毒素-抗毒素系统过表达潜伏感染实时定量PCR技术诊断　出处：《石河子大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:(1).构建北京、非北京基因型结核分枝杆菌、标准株(H37RV)的mazEF3、mazEF6、mazEF9基因的过表达菌株,观测其在低氧、营养缺乏条件下的生存能力,同时检测不同电转电压下过表达菌株相应基因的mRNA表达水平。(2).检测预测的7种细胞因子的mRNA的表达水平,探讨目的因子分子表达水平作为结核分枝杆菌潜伏感染诊断的生物标志物的可能性。方法:(1).利用大肠杆菌-结核分枝杆菌穿梭表达质粒pMV361,构建出重组质粒pMV361-mazEF3、pMV361-mazEF6、pMV361-mazEF9;电穿孔将重组质粒导入北京基因型、非北京基因型结核分枝杆菌中。在卡那霉素培养基上初步筛选,提取阳性菌株的质粒进行PCR、双酶切,送公司测序,验证构建是否成功;检测不同菌株在不同生长条件下的生长情况。实时荧光定量PCR技术检测不同电转电压下mazEF3的mRNA表达水平,寻找构建mazEF3过表达菌株的最佳电转电压。(2).健康对照组64人,活动性肺结核50人,结核分枝杆菌潜伏感染60人,实时荧光定量PCR(qRT-PCR)检测结核特异性抗原刺激的外周血中TNF-α、IFN-γ、IL-2、IL-10、IFI35、IP10及Foxp3的mRNA表达水平。结果:(1).成功构建了H37RV、北京、非北京基因型的结核分枝杆菌的mazEF3、mazEF6、mazEF9过表达菌株。北京基因型的结核分枝杆菌在正常和应激环境下,生长状态均好于非北京基因型结核分枝杆菌。实时荧光定量检测,电转电压为2300V时,构建的过表达菌株mazEF3的mRNA表达量最高。(2).7种因子相对表达量在活动性肺结核组为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,潜伏感染组分别为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,健康组分别为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。潜伏感染组与健康组相比7种细胞因子的mRNA表达水平,差异均有统计学意义(P0.01),相应细胞因子检测的敏感性、特异性分别为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%;潜伏感染组和活动性结核组比较除IFI35因子外,其余6种细胞因子的mRNA表达水平差异均有统计学意义(P0.05),相应细胞因子检测的特异性、敏感性分别为90.6%、80%,88.6%、92.7%,91.9%、86.2%,57.6%、60%,50%、58%,76%、72.2%和66.7%、67.1%;健康对照组和活动性结核组相比较,7种细胞因子的mRNA表达水平差异均有统计学意义(P0.01),相应细胞因子检测的敏感性、特异性分别为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%。结核病密切接触者潜伏感染率为48.97%,健康组潜伏感染率为36%。结论:(1).成功构建了北京、非北京基因型的mazEF3、mazEF6、mazEF9过表达菌株,2300V是mazEF3的最佳电转电压。(2).结核病患者血清TNF-α、IFN-γ、IL-2 mRNA水平升高,以潜伏感染者升高更显著,因此可作为诊断结核分枝杆菌潜伏感染的生物标志物。
[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- 伪 IFN- 纬 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 卤0.09 in active pulmonary tuberculosis group. 4.94 卤0.45U 150 卤17.82U 2.02 卤0.55U 9.53 卤5.96U 5.56 卤0.97U 1.24 卤0.17. The latent infection group was 3.2 卤0.61g 10.04 卤1.89 卤1.89 卤21.89 卤10.25 卤29.17 卤11.19, respectively. 41.35 卤11.46 卤2.14 卤0.67 in the control group and 1.2 卤0.08 卤2.92 卤0.03 卤53 卤17.82 卤1.48 卤0.17 in the healthy group, respectively. 1.28 卤0.77 ~ 1.63 卤0.25 ~ 1.03 卤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- 伪 of patients with tuberculosis is TNF- 伪. The level of IL-2 mRNA in IFN- 纬 is higher than that in latent infection, so it can be used as a biomarker for the diagnosis of latent infection of Mycobacterium tuberculosis.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R378.911

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