【摘要】：目的：調(diào)查多重耐藥銅綠假單胞菌株中可移動性遺傳元件的分布情況,并以整合子為對象深入研究,探究整合子中不同可變區(qū)啟動子Pant對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種可移動遺傳元件的分布情況,并對可移動元件的組合形式進(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)含有無耐藥基因盒整合子和aadA2基因盒的重組質(zhì)粒pACYC-IN-AAD,通過表型篩選法測定其整合頻率；通過點(diǎn)突變法構(gòu)建含有不同可變區(qū)啟動子的重組質(zhì)粒,通過實(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種可移動遺傳元件相關(guān)遺傳標(biāo)記基因,4個(gè)種類的可移動性遺傳元件均有檢出。陽性率最高的為IS26(77.6%),其次為intI1(67%)。于國內(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)整合酶的整合子對耐藥基因盒的整合能力是無高表達(dá)整合酶整合子的9.41倍；不同可變區(qū)啟動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。可變區(qū)啟動子的啟動強(qiáng)度與下游的耐藥基因盒表達(dá)水平關(guān)系密切,啟動強(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)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R378

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 Ayseg ül Copur Cicek;Azer ?zad Düzgün;Aysegül Saral;Tuba Kayman;Zeynep Cizmeci;Pervin ?zlem Balci;Tuba Dal;Mehmet Firat;Ismail Tosun;Yasemin Ay Alitntop;Ahmet Caliskan;Yelda Yazici;Cemal Sandalli;;Detection of class 1 integron in Acinetobacter baumannii isolates collected from nine hospitals in Turkey[J];Asian Pacific Journal of Tropical Biomedicine;2013年09期

2 Reza Mirnejad;Sepideh Mostofi;Faramaz Masjedian;;Antibiotic resistance and carriage class 1 and 2 integrons in clinical isolates of Acinetobacter baumannii from Tehran,Iran[J];Asian Pacific Journal of Tropical Biomedicine;2013年02期

3 胡昌俊;朱艮苗;;銅綠假單胞菌Ⅰ類整合子陽性株對細(xì)菌耐藥的影響[J];重慶醫(yī)學(xué);2013年25期

4 王海生;扈瑞平;高玉敏;楊鐘;鄧秀玲;張建宇;武瑞兵;呼和巴特爾;;牛源大腸桿菌Ⅰ類整合子分子特征及其與耐藥性關(guān)系的研究[J];中國畜牧獸醫(yī);2014年09期

5 袁璐;劉紅波;鄒雅如;許歡;陳體;漆涌;伍勇;;細(xì)菌整合子調(diào)控機(jī)制中啟動子的作用研究進(jìn)展[J];臨床檢驗(yàn)雜志;2014年08期

6 姜波;魏取好;呂火祥;;尿液分離大腸埃希菌Ⅰ類整合子分子特征與耐藥性的關(guān)系研究[J];中華醫(yī)院感染學(xué)雜志;2014年01期

7 高昂;于紅;;產(chǎn)超廣譜β-內(nèi)酰胺酶細(xì)菌中I類整合子的研究進(jìn)展[J];微生物學(xué)通報(bào);2013年11期

8 林居純;舒剛;張輝建;曹三杰;文心田;;健康畜禽腸道大腸桿菌耐藥性及整合子-基因盒檢測[J];中國獸醫(yī)學(xué)報(bào);2014年01期

9 周宇石;李夢嬌;郭麗萍;滕浩波;于紅;;青島地區(qū)大腸埃希菌中I類整合子與耐藥性的分析[J];中國微生態(tài)學(xué)雜志;2014年05期

10 高昂;王昕凝;石志源;丁印;于紅;;青島地區(qū)產(chǎn)ESBLs菌株Ⅰ類整合子的分布與耐藥性分析[J];中國抗生素雜志;2014年04期

相關(guān)博士學(xué)位論文 前1條

1 孫光明;整合子介導(dǎo)銅綠假單胞菌獲得性耐藥機(jī)制的研究[D];江蘇大學(xué);2013年

相關(guān)碩士學(xué)位論文 前1條

1 黃長武;多重耐藥鮑曼不動桿菌整合子分型及流行病學(xué)研究[D];西南大學(xué);2014年

，

本文編號：2339662