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目的探讨基因毒性物质介导的DNA损伤引起分枝杆菌SOS反应的作用。方法选择耻垢分枝杆菌为分枝杆菌模式细菌,通过紫外线损伤试验检测耻垢分枝杆菌对紫外线的敏感性,通过2倍稀释法测定利福平和氧氟沙星的最小抑菌浓度(MIC)。分别用紫外线和次抑菌浓度(1/2MIC)抗生素处理耻垢分枝杆菌,在不同时间点收集菌液,以看家基因sigA为内参,采用实时定量PCR相对定量方法检测dnaE2、recA和recX等SOS基因表达水平的变化。结果紫外线暴露45s后耻垢分枝杆菌的存活率为50%,利福平和氧氟沙星的MIC分别是32μg/ml和1μg/ml。耻垢分枝杆菌dnaE2和recX等SOS基因表达水平在紫外线下暴露45s后即开始上升,recA的表达水平在紫外线暴露3h后显著上升。耻垢分枝杆菌dnaE2和recX基因表达水平在1/2MIC的利福平作用后1h或在1/2MIC的氧氟沙星作用0h后上升,recA的表达水平在1/2MIC的利福平或氧氟沙星作用3h时显著升高。结论紫外线、抗生素等基因毒性物质可以导致耻垢分枝杆菌DNA损伤及SOS基因表达水平上升。
Objective To investigate the role of genotoxic substances-induced DNA damage in mycobacterium SOS. Methods Mycobacterium smegmatis was selected as mycobacterium. The sensitivity of Mycobacterium smegmatis to UV light was tested by ultraviolet light damage test. The minimum inhibitory concentration (MIC) of rifampicin and ofloxacin ). Mycobacterium smegmatis was treated with UV and secondary bacteriostatic concentration (1 / 2MIC) antibiotics respectively, and bacterial liquid was collected at different time points. The housekeeping gene sigA was used as an internal control, and quantitative real-time PCR was used to detect dnaE2, recA and recX And other SOS gene expression changes. Results The survival rate of Mycobacterium smegmatis was 50% after UV exposure for 45s. The MICs of rifampin and ofloxacin were 32μg / ml and 1μg / ml, respectively. The levels of SOS gene in Mycobacterium smegmatis such as dnaE2 and recX began to increase after exposure to ultraviolet light for 45s, and the level of recA expression increased significantly 3h after UV exposure. The levels of mycobacterium smegmatis dnaE2 and recX mRNA increased 1 h after 1 / 2MIC of rifampin or 0 h after 1 / 2MIC ofloxacin, and the expression of recA was up to 1 / 2MIC of rifampin or Ofloxacin role 3h significantly increased. Conclusions UV, antibiotics and other genotoxic substances can cause the DNA damage of M. smegmatis and the increase of SOS gene expression.