發(fā)布時(shí)間：2018-11-18 09:34

【摘要】：目的：調(diào)查多重耐藥銅綠假單胞菌株中可移動(dòng)性遺傳元件的分布情況,并以整合子為對(duì)象深入研究,探究整合子中不同可變區(qū)啟動(dòng)子Pant對(duì)耐藥基因盒和整合酶的表達(dá)以及整合頻率的影響,了解啟動(dòng)子Pant在整合子介導(dǎo)的耐藥機(jī)制中的作用,從而為控制細(xì)菌廣泛耐藥和新型抗菌藥物的研發(fā)提供一定的理論基礎(chǔ)。 方法：從臨床標(biāo)本中分離并篩選出49株多重耐藥的銅綠假單胞菌,采用聚合酶鏈反應(yīng)(PCR)分析21種可移動(dòng)遺傳元件的分布情況,并對(duì)可移動(dòng)元件的組合形式進(jìn)行分析；構(gòu)建高表達(dá)整合酶基因的重組質(zhì)粒pET-INT和同時(shí)含有無(wú)耐藥基因盒整合子和aadA2基因盒的重組質(zhì)粒pACYC-IN-AAD,通過(guò)表型篩選法測(cè)定其整合頻率；通過(guò)點(diǎn)突變法構(gòu)建含有不同可變區(qū)啟動(dòng)子的重組質(zhì)粒,通過(guò)實(shí)時(shí)熒光定量PCR比較其aadA2基因和intI1基因的表達(dá)量。 結(jié)果：49株多重耐藥銅綠假單胞菌菌株中,共檢出11種可移動(dòng)遺傳元件相關(guān)遺傳標(biāo)記基因,4個(gè)種類的可移動(dòng)性遺傳元件均有檢出。陽(yáng)性率最高的為IS26(77.6%),其次為intI1(67%)。于國(guó)內(nèi)首次在多重耐藥銅綠假單胞菌中檢出traF基因；本研究成功構(gòu)建了整合子剪切和捕獲耐藥基因盒的體外模型,研究發(fā)現(xiàn),在BL21(DE3)菌株中,含有高表達(dá)整合酶的整合子對(duì)耐藥基因盒的整合能力是無(wú)高表達(dá)整合酶整合子的9.41倍；不同可變區(qū)啟動(dòng)子下游基因盒中基因的轉(zhuǎn)錄水平有顯著差異,4種質(zhì)粒的aadA2基因轉(zhuǎn)錄水平從高到低依次為：MU2pACYC-IN-AADMU3MU1,最高者是最低者的114.7倍。 結(jié)論：可移動(dòng)遺傳元件在銅綠假單胞菌株中廣泛存在,插入序列和整合子在其耐藥基因水平傳播中起主要的作用�？勺儏^(qū)啟動(dòng)子的啟動(dòng)強(qiáng)度與下游的耐藥基因盒表達(dá)水平關(guān)系密切,啟動(dòng)強(qiáng)度越高,基因盒表達(dá)越強(qiáng),反之亦然。圖14幅,表14個(gè),參考文獻(xiàn)42篇。
[Abstract]:Objective: to investigate the distribution of transportable genetic elements in multidrug resistant Pseudomonas aeruginosa strains and to study the integron. To explore the effect of different variable region promoter Pant on the expression and integration frequency of drug resistance gene box and integrase in integron, and to understand the role of promoter Pant in integron mediated drug resistance mechanism. It provides a theoretical basis for the control of bacterial extensive drug resistance and the development of new antimicrobial agents. Methods: 49 strains of multidrug resistant Pseudomonas aeruginosa were isolated and screened from clinical specimens. The distribution of 21 kinds of movable genetic elements were analyzed by polymerase chain reaction (PCR). The frequency of integration was determined by phenotypic screening method in the construction of recombinant plasmid pET-INT with high expression of integrase gene and the recombinant plasmid pACYC-IN-AAD, containing both cassette integron and aadA2 gene box. The recombinant plasmids containing different variable region promoters were constructed by point mutation method. The expression levels of aadA2 gene and intI1 gene were compared by real-time fluorescence quantitative PCR. Results: among 49 strains of multidrug resistant Pseudomonas aeruginosa, 11 kinds of mobile genetic elements were detected, and 4 kinds of mobile elements were detected. The highest positive rate was IS26 (77.6%), followed by intI1 (67%). TraF gene was first detected in multidrug resistant Pseudomonas aeruginosa in China. In this study, we successfully constructed an in vitro model of integron shearing and capturing drug resistance gene cassette. It was found that in BL21 (DE3) strain, The integration ability of integron containing high expression integrase to drug resistant gene box was 9.41 times as high as that of non-high expression integrase integron. The transcriptional level of aadA2 gene in the cassette of different variable region promoters was significantly different. The transcription level of aadA2 gene in the four plasmids was 114.7 times higher than that in the lowest one. Conclusion: mobile genetic elements are widely present in Pseudomonas aeruginosa. Insertion sequences and integrons play a major role in the horizontal transmission of drug resistance genes. The priming intensity of variable region promoter was closely related to the expression level of drug resistance gene box downstream. The higher the starting intensity, the stronger the gene box expression, and vice versa. There are 14 figures, 14 tables and 42 references.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R378

【共引文獻(xiàn)】

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相關(guān)博士學(xué)位論文 前1條

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相關(guān)碩士學(xué)位論文 前1條

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本文編號(hào)：2339662