siHybrids技术沉默铜绿假单胞菌外排泵mexB基因体外效应研究

发布时间：2018-02-12 09:08

  本文关键词： siHybrids技术 RNAi 铜绿假单胞菌 外排泵 real-timePCR MIC　出处：《华中科技大学》2010年硕士论文　论文类型：学位论文

【摘要】：目的:研究siHybrids技术对铜绿假单胞菌PAO1外排泵mexB基因体外沉默效应。 方法:1.针对铜绿假单胞菌野生株PAO1外排泵mexB基因设计并合成3条特异性siHybrids分子和一条阴性对照siHybrids分子。 2.在分子浓度为50nM下,分别以合成设计的siHybrids分子干扰铜绿假单胞菌PAO1,并设实验组为铜绿假单胞菌PAO1空白对照组,阴性对照组scambl(esc)-001,干预组siHybrids(si)-001,siHybrids(si)-002及siHybrids(si)-003。 3.分别在干预12小时后及24小时后采用实时荧光定量PCR检测各实验组中外排泵靶基因mexB[1]基因mRNA表达水平。 4.进一步采用Mueller-Hinton倍比稀释法检测50nM浓度的siHybrids分子对铜绿假单胞菌PAO1与氯霉素(CP),红霉素(EM),左氧氟沙星(L-OFLX)干预作用前后的MIC(最小抑菌浓度). 结果:1.不同siHybrids分子干预PAO1 12小时后,mexB基因mRNA的表达量无明显差异性。 2.干预24小时后,mexB基因mRNA的表达量干预组(si-001,si-002,si-003)比空白对照组,阴性对照组(sc-001)有明显下降。 3.对比干预12小时,24小时后mexB基因mRNA表达量,可以发现空白对照组,阴性对照组(sc-001)mRNA的表达量成上升趋势,而干预组(si-001,si-002,si-003)mexB基因mRNA表达量均呈下降趋势。 4.siHybrids分子在干预24小时前后氯霉素(CP),红霉素(EM),左氧氟沙星(L-OFLX)的MIC无明显差异性 结论:在mRNA的表达水平上, siHybrids分子能体外干预铜绿假单胞菌PAO1 mexB基因mRNA的表达,此种沉默作用呈现时间依赖性,且在24小时能有效地发挥干预作用。
[Abstract]:Aim: to study the silencing effect of siHybrids on mexB gene of Pseudomonas aeruginosa PAO1 efflux pump in vitro. Methods 1. Three specific siHybrids molecules and one negative control siHybrids molecule were designed and synthesized from the mexB gene of the wild strain of Pseudomonas aeruginosa PAO1 efflux pump. 2. At the molecular concentration of 50 nm, the designed siHybrids molecule was used to interfere with Pseudomonas aeruginosa PAO1, and the experimental group was divided into two groups: PAO1 blank control group, negative control group scamblesc-001, intervention group siHybridssi-001 and siHybridges sisi-002 and siHybridssisi-003. 3. After 12 hours and 24 hours of intervention, real-time quantitative PCR was used to detect the expression of mexB [1] gene in each experimental group. 4. Mueller-Hinton dilution method was used to detect the minimal inhibitory concentration (MEC) of 50nM siHybrids on Pseudomonas aeruginosa PAO1 and chloramphenicol, erythromycin Erythromycin and levofloxacin L-OFLX before and after the intervention. Results 1. There was no significant difference in the expression of mRNA of mexB gene in PAO1 treated with different siHybrids molecules for 12 hours. 2.After 24 hours of intervention, the mRNA expression of mexB gene in the intervention group was significantly lower than that in the blank control group and the negative control group. 3. Comparing the mRNA expression of mexB gene after 12 hours and 24 hours of intervention, it was found that in blank control group, the expression of sc-001 mRNA in negative control group showed an upward trend, while in intervention group, the mRNA expression of si-001si-002si-003mexB gene showed a downward trend. 4. The MIC of hybrids molecules before and after 24 hours of intervention were not significantly different from those of chloramphenicol, erythromycin and levofloxacin L-OFLX. Conclusion: at the level of mRNA expression, siHybrids molecule can interfere with the expression of PAO1 mexB gene mRNA of Pseudomonas aeruginosa in vitro, and the silencing effect is time dependent, and it can play an effective role in 24 hours.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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