迟缓爱德华氏菌两种保护性抗原及AcrAB耐药系统的分析

IOCAS-IR > 海洋环流与波动重点实验室

	迟缓爱德华氏菌两种保护性抗原及AcrAB耐药系统的分析
其他题名	Analysis of two protective antigens and the AcrAB multidrug resistance system of Edwardsiella tarda
	侯进慧
学位类型	博士
	2008-06-07
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	迟缓爱德华氏菌保护性抗原 Acrab 多药耐药性
摘要	迟缓爱德华氏菌是危害水产养殖业发展的重要病原菌之一，因而其免疫防治研究具有重要意义。论文分析了9种具有保护潜能的迟缓爱德华氏菌蛋白，经过牙鲆免疫保护实验，筛选出EseD和Et18两种有显著性保护效应的抗原。为了提高其保护效应，论文使用基因工程技术将这两种抗原融合到一起，构建重组融合蛋白EEH。结果表明，融合蛋白EEH保护效应较EseD和Et18分别免疫时有所提高。ELISA和Western blotting 结果显示，三种蛋白都能诱导牙鲆产生特异抗体。这些研究为开发迟缓爱德华氏菌疫苗提供了理论基础。论文克隆分析了迟缓爱德华氏菌AcrAB耐药系统，采用定点突变确定了acrAB、acrR的启动子序列和AcrR在acrAB启动子的结合位点。启动子分析显示，AcrR对acrAB启动子有300倍抑制效应, 对acrR启动子有3倍抑制效应。定点突变显示，K39和R45对AcrR功能具重要性；缺失突变表明，N端205个氨基酸残基是其功能必需。实验筛选出Acriflavine、Ethidium Bromide、Methyl Viologen、Sodium Dodecyl Sulfate等四种AcrR诱导物。分析AcrR过量表达菌株结果显示，其耐药性、生长状况和毒力水平较阴性对照组降低。这些研究加深了我们对迟缓爱德华氏菌耐药机制及其与毒力关系的了解。
其他摘要	Edwardsiella tarda is an important pathogenic bacterium in aquaculture, so its immune protection is of significance. In this study, 9 potential protective proteins of E. tarda were analyzed for the effect of immune protection by using Japanese flounder as animal models. The results showed that two of these proteins, EseD and Et18, exhibited significant protection. For the improvement of their protective efficiency, the two antigens were fused together via genentic engineering techniques and resulted in a hybrid protein EEH. Vaccination results revealed that the protective efficiency of EEH is higher than both EseD and Et18. ELISA and Western blotting analysis showed that the three recombinant proteins caused the production of significant levels of specific antibodies in Japanese flounder. This study provides theoretical basis for the development of E. tarda vaccines. In addition, the E. tarda AcrAB multidrug resistance system was cloned in this study. Sited-directed mutation was used to determine the promoter sequences of acrAB and acrR and the AcrR binding site. Promoter activity analysis demonstrated that the presence of AcrR casued 300-fold reduction in the activity of the acrAB promoter and 3-fold reduction in the activity of the acrR promoter. Sited-directed mutations showed that K39 and R45 are important for AcrR activity; deletion analysis results revealed that the N terminal 205 amino acid residues are essential to the function of AcrR. Acriflavine, Ethidium Bromide, Methyl Viologen and Sodium Dodecyl Sulfate are found to be the inductors of AcrR. Analysis of the acrR-overexpressing strain demonstrated that its drug resistance level, growth condition and virulence are lower than the control group. This study improves the understanding of multidrug resistance mechanism of E. tarda as well as its relevance to virulence.
页数	102
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/695
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	侯进慧. 迟缓爱德华氏菌两种保护性抗原及AcrAB耐药系统的分析[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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