【摘要】： 銅綠假單胞菌(Pseudomonas aeruginasa,PA)是一種革蘭氏陰性細(xì)菌,是醫(yī)院感染的三大致病菌之一,能夠引起致命性的急慢性感染。銅綠假單胞菌具有多重耐藥機(jī)制,并能在貧營(yíng)養(yǎng)、重污染等條件下抵抗脅迫生存,持久存活于肺纖維囊腫(Cystic Fibrosis,CF)病人體內(nèi)。在治療銅綠假單胞菌感染的過(guò)程中,有少量菌體在脅迫條件下存活于人體內(nèi),在一定的條件下再次引起感染,使得慢性病人長(zhǎng)期反復(fù)感染,難以治愈。因此研究PA的持久生存機(jī)制,在此基礎(chǔ)上開(kāi)發(fā)可以有效阻斷與病原菌持久存活相關(guān)的路徑,對(duì)于有效控制臨床上銅綠假單胞菌的長(zhǎng)期感染,打破傳統(tǒng)抗生素易于產(chǎn)生耐藥性的局限性具有重要意義。 本研究對(duì)以luxCDABE為報(bào)道子的銅綠假單胞菌PAO1隨機(jī)啟動(dòng)子庫(kù)(約3456個(gè)克隆)進(jìn)行篩選,尋找能夠在脅迫條件下引起持久發(fā)光的啟動(dòng)子,通過(guò)測(cè)序、比對(duì),確定這些啟動(dòng)子相應(yīng)的基因位置。挑選出其中具有長(zhǎng)期條件下表達(dá)典型的基因進(jìn)行敲除。對(duì)突變體與野生型菌株進(jìn)行比較:在脅迫條件下長(zhǎng)期培養(yǎng)并通過(guò)菌落形成單位(CFU)測(cè)其各自的存活率;對(duì)野生型與突變株進(jìn)行共培養(yǎng),讓其在競(jìng)爭(zhēng)狀態(tài)下長(zhǎng)期培養(yǎng),然后分別測(cè)定其存活率;設(shè)計(jì)長(zhǎng)時(shí)間培養(yǎng)的高滲透與低pH的脅迫條件來(lái)測(cè)定突變株在外加脅迫條件下的適應(yīng)性。 實(shí)驗(yàn)結(jié)果表明,從銅綠假單胞菌PAO1隨機(jī)啟動(dòng)子庫(kù)中篩選出157株在脅迫條件下長(zhǎng)期表達(dá)的菌株,其啟動(dòng)子對(duì)應(yīng)的基因被假定與PAO脅迫條件下生存相關(guān)。通過(guò)基因比對(duì),最后確定了45個(gè)與持久生存相關(guān)的基因,其中14個(gè)是推測(cè)生物合成代謝相關(guān)基因,5個(gè)是假定的編碼轉(zhuǎn)錄調(diào)節(jié)子,4個(gè)是假定的轉(zhuǎn)運(yùn)蛋白,另有4個(gè)已知基因和18個(gè)功能完全未知的基因。挑選其中7個(gè)(PA0423、PA1216、PA2827、PA0550、PA0256、PA0057、PA4578)重復(fù)性高且在在持久存活過(guò)程中表達(dá)明顯的基因作為實(shí)驗(yàn)的目的基因重點(diǎn)研究,利用基因敲除技術(shù)分別使目的基因功能缺失,得到7株突變株。通過(guò)對(duì)比實(shí)驗(yàn),結(jié)果表明:這七個(gè)基因均與菌體持久生長(zhǎng)相關(guān),其中五個(gè)基因(PA1216,PA0423,PA2827,PA0057,PA4578)在持久生存中起到重要作用,這些基因功能的缺失,造成細(xì)菌存活率明顯下降。本研究結(jié)果初步闡明了這七個(gè)相關(guān)基因和銅綠假單胞菌在脅迫條件下持久存活的關(guān)系,但其作用機(jī)理還需進(jìn)一步深入研究。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas) is a gram-negative bacteria, one of the three major nosocomial infections, which can cause fatal acute and chronic infections. Pseudomonas aeruginosa has the mechanism of multidrug resistance, and can survive under the condition of poor nutrition and heavy pollution, and can survive in patients with Cystic Fibrosis CF for a long time. In the course of treatment of Pseudomonas aeruginosa infection, a small number of bacteria survived in the human body under stress, and caused the infection again under certain conditions, which made chronic patients repeatedly infected for a long time and difficult to cure. Therefore, by studying the persistent survival mechanism of PA, we can effectively block the pathway related to the persistent survival of pathogenic bacteria and control the long-term infection of Pseudomonas aeruginosa clinically. It is of great significance to break the limitation that traditional antibiotics are prone to drug resistance. In this study, the random promoter library of Pseudomonas aeruginosa PAO1 (about 3456 clones) was screened with luxCDABE as reporter. The corresponding gene location of these promoters was determined. The genes with long term expression were selected for knockout. The mutants were compared with wild-type strains. The survival rate was measured by colony forming unit (CFU). The wild type and mutant were co-cultured for a long time under competitive condition. Then the survival rate of the mutant was determined, and the adaptability of the mutant under external stress was determined by designing the stress conditions of high osmotic and low pH for a long time. The results showed that 157 strains of Pseudomonas aeruginosa PAO1 promoter library were screened for long term expression under stress, and the corresponding gene of the promoter was assumed to be related to the survival of Pseudomonas aeruginosa under PAO stress. Through gene alignment, 45 genes related to persistent survival were identified, of which 14 were conjectural biosynthesis related genes, 5 were assumed transcriptional regulators, and 4 were assumed transporters. There were also 4 known genes and 18 completely unknown genes. Seven of them (PA0423, PA1216, PA2827, PA0550, PA0256, PA0057, PA4578) were selected as the key genes of the experiment. Seven mutants were obtained by using gene knockout technique. The results of comparative experiments showed that the seven genes were related to the sustained growth of bacteria, and five of them (PA1216 / PA0423 / PA2827PA0057 / PA4578) played an important role in the survival of the bacteria. The absence of these genes resulted in a significant decrease in the survival rate of bacteria. The relationship between the seven genes and the survival of Pseudomonas aeruginosa under stress was preliminarily clarified, but the mechanism of these seven genes should be further studied.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R378

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