本文選題：銅綠假單胞菌 + 轉(zhuǎn)座突變��； 參考：《西北大學(xué)》2007年碩士論文

【摘要】： 銅綠假單胞菌(Pseudomonas aerugionsa)是一種機(jī)會(huì)致病菌，它可以在人群中引起嚴(yán)重的急性和慢性感染，是病人在醫(yī)院期間發(fā)生感染的第三大致病菌。雖然某些抗生素對(duì)銅綠假單胞菌具有一定的治療作用，但同時(shí)，銅綠假單胞菌有一個(gè)重要特性是它對(duì)許多抗生素具有很高的內(nèi)在抗藥性，這使其成為臨床上難以治療的病原菌。研究銅綠假單胞菌抗藥性產(chǎn)生的機(jī)理、研究其與致病性相關(guān)的基因，對(duì)于了解其致病機(jī)理和控制其感染極為重要。 外排泵對(duì)抗生素的外排作用是細(xì)菌抗藥性的主要原因之一。銅綠假單胞菌中有12個(gè)已知和假定的外排泵基因簇。czcCBA基因簇編碼其中一個(gè)RND外排泵。本實(shí)驗(yàn)對(duì)這一RND泵的基因調(diào)節(jié)進(jìn)行了研究，在重金屬鹽ZnCl_2存在情況下，用隨機(jī)突變的方法篩選了基因組中czcCBA基因的調(diào)節(jié)子。經(jīng)過測(cè)試篩選大約20000個(gè)克隆，得到了7個(gè)對(duì)czcCBA基因表達(dá)有影響的突變體。這些突變體與野生型相比，czcCBA表達(dá)量發(fā)生了很大的變化，在有250nM Zn~(2+)存在的條件下，一個(gè)突變體中的表達(dá)量比原來提高了5倍，另外6個(gè)突變體幾乎沒有發(fā)光，即表達(dá)消失。經(jīng)重組敲除實(shí)驗(yàn)及互補(bǔ)實(shí)驗(yàn)驗(yàn)證，這7個(gè)基因與czcCBA的表達(dá)調(diào)節(jié)有關(guān)。 先前有研究證明致病菌能夠捕獲真核宿主細(xì)胞以及其他細(xì)菌的基因，并整合為自身的基因，從而提高自身對(duì)宿主免疫系統(tǒng)的抗性，進(jìn)而提高致病性。蛋白酶抑制因子α_2巨球蛋白，能夠捕獲致病菌成功侵襲所需的攻擊性蛋白酶，從而作為多細(xì)胞動(dòng)物抵御致病菌的主要防御機(jī)制之一。通過比較多細(xì)胞動(dòng)物和許多致病菌的基因序列，發(fā)現(xiàn)許多侵襲力更強(qiáng)，能使更高級(jí)真核細(xì)胞(動(dòng)物和植物)致病的病原菌具有α_2巨球蛋白的基因(Genome Biology 2004，5：R38)。對(duì)基因組的分析發(fā)現(xiàn)，在銅綠假單胞菌中也有α_2M基因的同源序列(即PA4489)。但國際上對(duì)銅綠假單胞菌中的這個(gè)基因的研究幾乎沒有。本研究的另一個(gè)內(nèi)容就是利用基因置換的方法對(duì)銅綠假單胞菌中這一可能的α_2M基因進(jìn)行了敲除。通過比較野生型和突變株對(duì)不同抗生素的抗性差異，發(fā)現(xiàn)突變體對(duì)多粘菌素的抗性降低，表明yfa-α_2M基因與銅綠假單胞菌對(duì)多粘菌素的抗性有關(guān)。
[Abstract]:Pseudomonas aerugionsa is a opportunistic pathogen, which can cause severe acute and chronic infection in the population. Although some antibiotics have some therapeutic effects on Pseudomonas aeruginosa, an important characteristic of Pseudomonas aeruginosa is that it has high intrinsic resistance to many antibiotics, which makes it difficult to be treated clinically. It is very important to study the mechanism of drug resistance of Pseudomonas aeruginosa and its pathogenicity related genes in order to understand the pathogenicity mechanism and control the infection of Pseudomonas aeruginosa. The efflux of efflux pump to antibiotics is one of the main reasons of bacterial drug resistance. In Pseudomonas aeruginosa, there are 12 known and assumed output-pump gene clusters. CzcCBA gene cluster encodes one of the RND efflux pumps. In this experiment, the gene regulation of RND pump was studied. In the presence of heavy metal salt ZnCl2, the regulator of czcCBA gene was screened by random mutation method. After testing and screening about 20 000 clones, 7 mutants with influence on czcCBA gene expression were obtained. The expression of czcCBA in one mutant was five times higher than that in the wild type, and the expression of the other 6 mutants was almost no luminescence, that is, the expression disappeared in the presence of 250 nm Zn ~ (2). The results of recombinant knockout and complementary experiments showed that these seven genes were related to the regulation of czcCBA expression. Previous studies have shown that pathogens can capture and integrate the genes of eukaryotic host cells and other bacteria into their own genes, thus increasing their resistance to the host immune system and thus enhancing their pathogenicity. The protease inhibitor 偽 2 macroglobulin can capture the aggressive protease required for the successful invasion of pathogenic bacteria, which is one of the main defense mechanisms of multicellular animals against pathogenic bacteria. By comparing the gene sequences of multicellular animals and many pathogenic bacteria, it was found that many pathogenic bacteria with stronger invasiveness could cause more advanced eukaryotic cells (animals and plants) with 偽 2 macroglobulin gene (Genome Biology 2004 5: R38). The genome analysis showed that the homologous sequence of 偽 -lipo2m gene (PA4489) was also found in Pseudomonas aeruginosa. But there is little international research on this gene in Pseudomonas aeruginosa. Another aspect of this study was to knockout this possible 偽 -S 2M gene in Pseudomonas aeruginosa by gene replacement. By comparing the resistance of wild type and mutant to different antibiotics, it was found that the resistance of mutants to polymyxin decreased, which indicated that yfa- 偽 _ 2 M gene was related to the resistance of Pseudomonas aeruginosa to polymyxin.
【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】：2007
【分類號(hào)】：R378

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