devR基因突变对结核分枝杆菌细胞毒性的影响
发布时间：2019-03-14 20:15
【摘要】：结核病的病死率仅次于HIV的感染,已经严重威胁到了全人类的生存和健康。我国作为结核病负担大国,结核分枝杆菌的耐药问题也日渐突出,因此研究结核分枝杆菌的耐药和基因突变机制,对于疾病的防控意义重大。课题组前期研究发现结核分枝杆菌双组份系统的dev R(反应调节子编码基因)在临床耐药菌株中出现不同程度的升高表达现象,并利用同源重组方法构建了dev R基因敲除的卡介苗菌株。本课题在前期工作基础上,构建了乙酰胺启动子诱导表达dev R的回补菌株,并通过验证dev R对巨噬细胞和上皮细胞的毒性作用,进一步探索了dev R缺失导致的休眠障碍与结核分枝杆菌细胞毒性的关系。一、dev R基因回补菌株构建及鉴定1.构建由乙酰胺启动子诱导表达dev R的重组BCG菌株利用PJV53质粒和Pcherry 3质粒及目的基因,构建乙酰胺启动子启动表达dev R的重组载体,电转化入dev R敲除的BCG菌株感受态中,经PCR、双酶切和测序鉴定正确后,命名此重组菌株为dev R回补菌株(Pcherry3-Acetamindase-dev R)。2.重组BCG菌株Pcherry3-Acetamindase-dev R生长特性的观察将BCG野生菌株、dev R敲除菌株和dev R回补菌株分别培养至对数生长期即OD600=0.6时,调整菌量至105 CFU/m L,以菌液和培养基比例为1:50接种至含有各自抗生素抗性的7H9液体培养基中培养,分别在第1-14 d测定OD600值,绘制细菌生长曲线。结果显示,dev R敲除菌株在早期(2~10 d)生长较BCG野生菌株明显加快(P0.01);dev R回补菌株和BCG野生菌株之间的生长无显著性差异(P0.05)。3.三种菌株对异烟肼和利福平的耐药发生率检测将BCG野生菌株、dev R敲除菌株和dev R回补菌株以1个Mc Farland浓度接种到分别含有10倍MIC浓度的异烟肼和利福平的7H10固体培养基上培养28天,计数培养基上生长出的菌落数,得到耐药发生率。结果显示,三种菌株对异烟肼和利福平的耐药发生率均无显著性差异(P0.05)。二、三种菌株分别感染上皮细胞A549和巨噬细胞Raw264.7的细胞毒性检测三种菌株均以感染复数(MOI)等于10∶1分别感染上皮细胞A549巨噬细胞Raw264.7,分别采用流式细胞仪和酶联免疫吸附试验于0、24、48、72 h测定细胞凋亡率和乳酸脱氢酶(LDH)释放水平;采用RT-PCR方法测定凋亡相关基因bcl-2和bad的表达。细胞感染试验结果显示,dev R敲除菌株感染A549细胞和Raw264.7细胞后,细胞的凋亡和坏死效应明显增加,相较于BCG野生菌株和dev R回补菌株差异显著(P0.05);三种菌株感染A549细胞均导致凋亡相关基因bcl-2表达升高,并且dev R敲除菌株感染组明显高于BCG和dev R回补菌株感染组(P0.05);三种菌株感染Raw264.7细胞均导致凋亡相关基因bad表达升高,并且dev R敲除菌株感染组明显高于BCG野生菌株和dev R回补菌株感染组(P0.05)。本研究初步证明了dev R敲除的BCG菌株毒力明显增强。
[Abstract]:The mortality rate of tuberculosis, second only to HIV infection, has seriously threatened the survival and health of all mankind. As a major burden of tuberculosis in China, the problem of drug resistance of Mycobacterium tuberculosis is becoming more and more prominent. Therefore, it is of great significance to study the mechanism of drug resistance and gene mutation of Mycobacterium tuberculosis in order to prevent and control the disease. Previous studies in our team found that the dev R (response regulator gene of Mycobacterium tuberculosis bicomponent system was expressed in different degrees in clinical drug-resistant strains, and the expression level of the two-component system of Mycobacterium tuberculosis was significantly higher than that of the control group. The Bacillus Calmette-Guerin (BCG) strain with dev R gene knockout was constructed by homologous recombination. On the basis of previous work, a complement strain of dev-R induced by acetamide promoter was constructed, and the toxicity of dev-R on macrophages and epithelial cells was verified. The relationship between dormancy disorder caused by dev R deletion and cytotoxicity of Mycobacterium tuberculosis was further investigated. Construction and Identification of a, dev R Gene complementing strain The recombinant BCG strain expressing dev R induced by acetamide promoter was constructed by using PJV53 plasmid, Pcherry 3 plasmid and target gene to construct the recombinant vector of dev R expression initiated by acetamide promoter. The recombinant vector was transformed into the competent state of dev R knockout strain BCG by electroporation, and the recombinant vector was transformed into the competent state of dev R knockout strain by PCR,. After being identified by double enzyme digestion and sequencing, the recombinant strain was named as dev R complementary strain (Pcherry3-Acetamindase-dev R). 2.) Observation on the growth characteristics of recombinant BCG strain Pcherry3-Acetamindase-dev R the wild BCG strain, dev R knockout strain and dev R supplementary strain were cultured to the logarithmic growth period (OD600=0.6) respectively, and the bacterial quantity was adjusted to 105 CFU/m / L. The bacteria were cultured in 7H9 liquid medium containing antibiotic resistance at the ratio of 1:50 to 1:50. OD 600 was measured on the 1st-14th day, and the growth curve of the bacteria was drawn. The results showed that the growth of, dev R knockout strain was significantly faster than that of BCG wild strain (P0.01) (P0.01). There was no significant difference in the growth of); dev R knockout strain and BCG wild strain (P0.05). Detection of resistance rates of three strains to isoniazid and rifampicin in wild strains of BCG The dev R knockout strain and the dev R complement strain were inoculated on 7H10 solid medium containing 10 times MIC concentration of isoniazid and rifampicin respectively in a Mc Farland concentration for 28 days. The number of colonies grown on the medium was counted and the incidence of drug resistance was obtained. The results showed that there was no significant difference in the rates of resistance to isoniazid and rifampicin among the three strains (P0.05). Cytotoxicity of three strains infected with A549 and Raw264.7 respectively; all of the three strains infected A549 cells with multiple numbers of (MOI) equal to 10:1, respectively, infected with Raw264.7, in epithelial cells A549 and macrophages, respectively. Cell apoptosis rate and lactate dehydrogenase (LDH) release were measured by flow cytometry and enzyme-linked immunosorbent assay (Elisa) at 0,24,48,72 h, respectively. The expression of apoptosis-related genes bcl-2 and bad was determined by RT-PCR. The results of cell infection test showed that the apoptosis and necrosis of A549 cells and Raw264.7 cells infected by, dev R knockout strain increased significantly, compared with wild strain BCG and dev R strain (P0.05). The expression of apoptosis-related gene bcl-2 was increased in A549 cells infected by three kinds of strains, and the expression of dev R knockout strain was significantly higher in dev R knockout group than that in BCG and dev R backfill group (P0.05). The expression of apoptosis-related gene bad was increased in Raw264.7 cells infected by three strains, and the expression of dev R knockout strain was significantly higher in dev R knockout group than that in BCG wild strain and dev R backfill strain group (P0.05). The results showed that the virulence of dev R knockout strain BCG was significantly enhanced.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
本文编号：2440333
[Abstract]:The mortality rate of tuberculosis, second only to HIV infection, has seriously threatened the survival and health of all mankind. As a major burden of tuberculosis in China, the problem of drug resistance of Mycobacterium tuberculosis is becoming more and more prominent. Therefore, it is of great significance to study the mechanism of drug resistance and gene mutation of Mycobacterium tuberculosis in order to prevent and control the disease. Previous studies in our team found that the dev R (response regulator gene of Mycobacterium tuberculosis bicomponent system was expressed in different degrees in clinical drug-resistant strains, and the expression level of the two-component system of Mycobacterium tuberculosis was significantly higher than that of the control group. The Bacillus Calmette-Guerin (BCG) strain with dev R gene knockout was constructed by homologous recombination. On the basis of previous work, a complement strain of dev-R induced by acetamide promoter was constructed, and the toxicity of dev-R on macrophages and epithelial cells was verified. The relationship between dormancy disorder caused by dev R deletion and cytotoxicity of Mycobacterium tuberculosis was further investigated. Construction and Identification of a, dev R Gene complementing strain The recombinant BCG strain expressing dev R induced by acetamide promoter was constructed by using PJV53 plasmid, Pcherry 3 plasmid and target gene to construct the recombinant vector of dev R expression initiated by acetamide promoter. The recombinant vector was transformed into the competent state of dev R knockout strain BCG by electroporation, and the recombinant vector was transformed into the competent state of dev R knockout strain by PCR,. After being identified by double enzyme digestion and sequencing, the recombinant strain was named as dev R complementary strain (Pcherry3-Acetamindase-dev R). 2.) Observation on the growth characteristics of recombinant BCG strain Pcherry3-Acetamindase-dev R the wild BCG strain, dev R knockout strain and dev R supplementary strain were cultured to the logarithmic growth period (OD600=0.6) respectively, and the bacterial quantity was adjusted to 105 CFU/m / L. The bacteria were cultured in 7H9 liquid medium containing antibiotic resistance at the ratio of 1:50 to 1:50. OD 600 was measured on the 1st-14th day, and the growth curve of the bacteria was drawn. The results showed that the growth of, dev R knockout strain was significantly faster than that of BCG wild strain (P0.01) (P0.01). There was no significant difference in the growth of); dev R knockout strain and BCG wild strain (P0.05). Detection of resistance rates of three strains to isoniazid and rifampicin in wild strains of BCG The dev R knockout strain and the dev R complement strain were inoculated on 7H10 solid medium containing 10 times MIC concentration of isoniazid and rifampicin respectively in a Mc Farland concentration for 28 days. The number of colonies grown on the medium was counted and the incidence of drug resistance was obtained. The results showed that there was no significant difference in the rates of resistance to isoniazid and rifampicin among the three strains (P0.05). Cytotoxicity of three strains infected with A549 and Raw264.7 respectively; all of the three strains infected A549 cells with multiple numbers of (MOI) equal to 10:1, respectively, infected with Raw264.7, in epithelial cells A549 and macrophages, respectively. Cell apoptosis rate and lactate dehydrogenase (LDH) release were measured by flow cytometry and enzyme-linked immunosorbent assay (Elisa) at 0,24,48,72 h, respectively. The expression of apoptosis-related genes bcl-2 and bad was determined by RT-PCR. The results of cell infection test showed that the apoptosis and necrosis of A549 cells and Raw264.7 cells infected by, dev R knockout strain increased significantly, compared with wild strain BCG and dev R strain (P0.05). The expression of apoptosis-related gene bcl-2 was increased in A549 cells infected by three kinds of strains, and the expression of dev R knockout strain was significantly higher in dev R knockout group than that in BCG and dev R backfill group (P0.05). The expression of apoptosis-related gene bad was increased in Raw264.7 cells infected by three strains, and the expression of dev R knockout strain was significantly higher in dev R knockout group than that in BCG wild strain and dev R backfill strain group (P0.05). The results showed that the virulence of dev R knockout strain BCG was significantly enhanced.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
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1 旦正尖措;devR基因突变对结核分枝杆菌细胞毒性的影响[D];第四军医大学;2015年
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