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  • 學位論文

探討細菌aconitase的碳端部分蛋白對鎘離子抗性

The characterization of cadmium resistance mediated by C-terminal portion of bacterial aconitase gene

指導教授 : 蔡淦仁
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摘要


因為治療藥物的普及,一般民眾取得方便,造成了「藥物濫用」這個大問題,也導致許多微生物抗藥性的產生,最麻煩的是醫院院內的感染。在眾多的院內感染病源菌之中,MRSA (或稱ORSA,methicillin/oxacillin-resistant Staphylococcus aure)一直高居榜首之位。最近,我們實驗室也發現臨床分離具有bata-lactam抗性的MRSA,大約有百分之九十,同時具有對鎘毒性的抗性,探究產生的原因是MRSA具有cadA和cadB兩種主要產生抗性基因的質體。cadA已經被證實有抗鎘能力,cadB則是具有相當弱的抗性能力。根據這個發現指示,在抗生素的抗性和對重金屬的抗性間,有一些基因學上的關聯性。於是,我們大量篩選葡萄球菌的基因,看看是否有新的抗性基因存在。篩選的結果,我們找到一段基因,對於鎘離子具有高抵抗性,和cadA比較,幾乎不分軒輊。經過序列分析,發現這一段基因和E.coli之aconitase的C (碳)端相似,於是將發現的這段基因命名為cp-acnA。為了更進一步分析cp-acnA的特性,我們利用PCR的方式,產生cp-acnA這段基因,再將這段基因接合在pSE380和pBAD質體內,送入大腸桿菌體內作表達。根據本實驗結果顯示,Cp-acnA蛋白可以顯示在SDS-PAGE的膠片上,而具有這段基因的大腸桿菌,對於金屬離子Cd2+、Zn2+、Pb2+、Cu2+和Cu+等擁有相當程度的抵抗性,這一個能力並不會受到氫離子濃度梯度高低、溫度升降和glutathione還原酶消耗的影響。在抗生素抗性方面,這段基因也賦予大腸桿菌對Tetracycline的抗性。所以我們可以再次證明,抗生素的抗性與鎘離子的抗性的確有些相關性。

並列摘要


Inappropriate usages of antibiotics raised drug-resistant microbes become the nightmare in treatment of infectious diseases. Among them, the methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of hospital infections. Recently, we have found an approximately 90% of β-lactam resistant MRSAs are also conferred resistance to cadmium. The MRSA cadmium resistance has been demonstrated by harboring either cadA or cadB genes, or both. The cadA gene product encoded the major cadmium resistant phenomenon, and cadB gene product plays a minor role in the resistance. In order to establish the genetic linkages between the antibiotic and cadmium resistance, and to determine if there is any other cadmium resistant gene, we have performed this thesis research to screen bacterial nucleic acids throughout the staphylococcal genome. A DNA fragment with a very high cadmium resistance has been identified. DNA sequence analysis has shown that this DNA fragment is within the 3’-end of bacterial aconitase gene, called cp-acnA. In order to determine the cadmium resistance mediated by cp-acnA gene product, we have cloned and expressed this gene fragment in E. coli. Our results have shown that cp-acnA gene product revealed some protections to Cd2+/Zn2+/Pb2+ in RW3110 and Cu2+/Cu+ in LMG/DC194. The resistance mediated by cp-acnA gene did not change in different H+ concentrations, various temperatures and under GSH-depletion condition. Moreover, we also found that cp-acnA gene might also associate with the tetracycline resistance in E. coli. In this study, we have demonstrated that there are some links between the antibiotic and cadmium resistances.

參考文獻


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