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  本文選題：銅綠假單胞菌　切入點(diǎn)：生物被膜　出處：《華中科技大學(xué)》2012年碩士論文

【摘要】：【目的】利用RNA干擾技術(shù)（RNA interference，RNAi）抑制銅綠假單胞菌PBP-1b編碼基因mrcB的表達(dá)，，探討PBP-1b對(duì)藻酸鹽編碼基因algD產(chǎn)生的影響，以期為臨床生物被膜耐藥提供新的思路。 【方法】采用siRNA表達(dá)載體的方法，分別設(shè)計(jì)2對(duì)靶向mrcB基因shRNA序列，構(gòu)建pMF54mrcB shRNA干擾載體；將構(gòu)建好的干擾載體及空載分別電轉(zhuǎn)入銅綠假單胞菌中，RT-PCR篩選抑制效率高的干擾載體，并分別計(jì)算第24小時(shí)、第三天、第六天的抑制率；同時(shí)用RT-PCR的方法分別檢測(cè)PAO1標(biāo)準(zhǔn)株及轉(zhuǎn)化株中藻酸鹽編碼基因algD分別在這三個(gè)時(shí)間點(diǎn)內(nèi)的的表達(dá)情況，通過(guò)統(tǒng)計(jì)學(xué)處理觀察algD的表達(dá)量有無(wú)差異。 【結(jié)果】經(jīng)DNA測(cè)序鑒定，成功構(gòu)建pMF54mrcB shRNA1/pMF54mrcB shRNA2干擾載體；將pMF54mrcB shRNA1/pMF54mrcB shRNA2/pMF54電轉(zhuǎn)入銅綠假單胞菌中，分別與空白組對(duì)照，RT-PCR檢測(cè)第24小時(shí)mrcB表達(dá)量pMF54mrcB shRNA2有明顯統(tǒng)計(jì)學(xué)差異。計(jì)算其第24小時(shí)、第三天及第六天的抑制率分別為26%、41%、9%。單因素方差分析示標(biāo)準(zhǔn)株的algD基因在三個(gè)時(shí)間點(diǎn)表達(dá)的差異有統(tǒng)計(jì)學(xué)意義，第三天最高；轉(zhuǎn)化株的表達(dá)量，在這三個(gè)時(shí)間點(diǎn)都比較高，無(wú)統(tǒng)計(jì)學(xué)意義。在這三個(gè)時(shí)間點(diǎn)用T-檢驗(yàn)分別比較標(biāo)準(zhǔn)株及轉(zhuǎn)化株algD基因的表達(dá)量，第三天無(wú)統(tǒng)計(jì)學(xué)意義，P＞0.05。 【結(jié)論】 PAO1標(biāo)準(zhǔn)株藻酸鹽編碼基因algD在生物被膜形成的第三天表達(dá)量最高；基因沉默的轉(zhuǎn)化株其algD的表達(dá)量在這三個(gè)時(shí)間點(diǎn)明顯增高，且其增高的時(shí)間提前，維持時(shí)間長(zhǎng)；分別對(duì)標(biāo)準(zhǔn)株及轉(zhuǎn)化株在三個(gè)時(shí)間點(diǎn)的algD表達(dá)量進(jìn)行統(tǒng)計(jì)學(xué)處理，第三天表達(dá)量無(wú)統(tǒng)計(jì)學(xué)意義，可能與PAO1algD的表達(dá)量在第三天最高有關(guān)；PBP-1b與藻酸鹽產(chǎn)生關(guān)系的具體調(diào)控機(jī)制還不詳，有待于進(jìn)一步研究發(fā)現(xiàn)。
[Abstract]:[objective] RNA interference technique was used to inhibit the expression of PBP-1b encoding gene mrcB in Pseudomonas aeruginosa, and to explore the effect of PBP-1b on the production of alginate encoding gene algD in order to provide a new idea for clinical biofilm drug resistance. [methods] using siRNA expression vector, two pairs of shRNA sequences targeting mrcB gene were designed to construct pMF54mrcB shRNA interference vector, and the constructed interference vector and no-load interference vector were respectively transferred into Pseudomonas aeruginosa by RT-PCR to screen the interference vectors with high inhibition efficiency. The inhibition rates of 24 hours, 3 days and 6 days were calculated, and the expression of alginate encoding gene algD in PAO1 standard strain and transformed strain were detected by RT-PCR. The expression of algD was observed by statistical analysis. [results] pMF54mrcB shRNA1/pMF54mrcB shRNA2 interference vector was successfully constructed by DNA sequencing, pMF54mrcB shRNA1/pMF54mrcB shRNA2/pMF54 was electrotransferred into Pseudomonas aeruginosa, and there was significant difference between pMF54mrcB shRNA1/pMF54mrcB shRNA2/pMF54 and control group in 24 h mrcB expression of pMF54mrcB shRNA2. The inhibition rates on the third day and the sixth day were 260.The single factor variance analysis showed that the algD gene expression of the standard strain was significantly different at the three time points, the highest on the third day; the expression level of the transformed strain was relatively high at these three time points. T- test was used to compare the expression of algD gene between the standard strain and the transformed strain at the three time points, but there was no significant difference between them on the third day (P > 0.05). [conclusion] the expression of alginate coding gene algD in PAO1 standard strain was the highest on the third day after the formation of biofilm, and the expression of algD in the transformed strain of gene silencing increased significantly at these three time points, and the time of increase was earlier and the time of maintenance was long. The algD expression of the standard strain and the transformed strain at three time points were statistically analyzed, but there was no statistical significance on the third day. The regulation mechanism of the relationship between PBP-1b and alginate production is unknown, which may be related to the highest expression of PAO1algD on the third day, which needs further study and discovery.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R378

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本文編號(hào)：1692570