本文關鍵詞： siHybrids技術 RNAi 銅綠假單胞菌 外排泵 real-timePCR MIC　出處：《華中科技大學》2010年碩士論文　論文類型：學位論文

【摘要】：目的:研究siHybrids技術對銅綠假單胞菌PAO1外排泵mexB基因體外沉默效應。 方法:1.針對銅綠假單胞菌野生株PAO1外排泵mexB基因設計并合成3條特異性siHybrids分子和一條陰性對照siHybrids分子。 2.在分子濃度為50nM下,分別以合成設計的siHybrids分子干擾銅綠假單胞菌PAO1,并設實驗組為銅綠假單胞菌PAO1空白對照組,陰性對照組scambl(esc)-001,干預組siHybrids(si)-001,siHybrids(si)-002及siHybrids(si)-003。 3.分別在干預12小時后及24小時后采用實時熒光定量PCR檢測各實驗組中外排泵靶基因mexB[1]基因mRNA表達水平。 4.進一步采用Mueller-Hinton倍比稀釋法檢測50nM濃度的siHybrids分子對銅綠假單胞菌PAO1與氯霉素(CP),紅霉素(EM),左氧氟沙星(L-OFLX)干預作用前后的MIC(最小抑菌濃度). 結果:1.不同siHybrids分子干預PAO1 12小時后,mexB基因mRNA的表達量無明顯差異性。 2.干預24小時后,mexB基因mRNA的表達量干預組(si-001,si-002,si-003)比空白對照組,陰性對照組(sc-001)有明顯下降。 3.對比干預12小時,24小時后mexB基因mRNA表達量,可以發(fā)現空白對照組,陰性對照組(sc-001)mRNA的表達量成上升趨勢,而干預組(si-001,si-002,si-003)mexB基因mRNA表達量均呈下降趨勢。 4.siHybrids分子在干預24小時前后氯霉素(CP),紅霉素(EM),左氧氟沙星(L-OFLX)的MIC無明顯差異性 結論:在mRNA的表達水平上, siHybrids分子能體外干預銅綠假單胞菌PAO1 mexB基因mRNA的表達,此種沉默作用呈現時間依賴性,且在24小時能有效地發(fā)揮干預作用。
[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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R378

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