迟缓爱德华氏菌铁调控蛋白与溶血素调控蛋白的分子生物学研究

IOCAS-IR > 海洋环流与波动重点实验室

	迟缓爱德华氏菌铁调控蛋白与溶血素调控蛋白的分子生物学研究
	王芳
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 Fur基因启动子定点突变功能域功能互补自我调控过量表达外膜蛋白溶血素 Gntr
摘要	爱德华氏菌病是海水养殖鱼类牙鲆（Japanese flounder）的一种常见细菌病。迟缓爱德华氏菌的致病相关因子有凝血素、溶血素、铁载体、粘附素及侵袭素等，其中调控铁载体的蛋白即是铁调控蛋白（Ferric uptake regulator）Fur。本论文首先用简并PCR及染色体步移的方法获得了迟缓爱德华氏菌fur基因(etfur)(GenBank 号EF197912)，发现其氨基酸序列与大肠杆菌的fur基因(ecfur)的氨基酸序列有大约90％的相似性。功能分析表明迟缓爱德华氏菌Fur（Etfur）能与大肠杆菌的Fur(Ecfur)功能互补；功能域分析发现Etfur的C92和C95为功能所必需，而E112K突变则增强FurEt的抑制功能；启动子活性分析发现Etfur具有自我调控功能，随之通过定点突变及引物延伸的方法确定了etfur的启动子。为了研究Etfur的调控干扰作用，我们构建了Etfur过量表达菌株TX1/pJRTF,发现该菌株的生长速度明显比野生型菌株慢，而且其外膜蛋白的表达与野生型相比有明显差异；随后我们提取了差异蛋白，用反免疫方法筛选差异表达基因。进一步的毒力研究表明Etfur过量表达能使细菌毒力显著下降。分析迟缓爱德华氏菌溶血素基因(ethB)启动子活性发现Etfur能够调控ethB的表达。最后，我们构建了一个调控抑制子筛选系统，并由此筛选到另一个ethB调控因子EthR,该调控因子属于GntR调控蛋白家族；实验分析表明EthR是一个比Fur效应更强的调控因子。
其他摘要	Edwardsiellosis is a common bacterial disease in mari-culture Japanese flounders. Some potential virulence factors of Edwardsiella tarda have been identified. These include siderophores, haemolysins, cell-adhesion and invasion factors. The fur gene (GeneBank accession no. EF197912) was cloned by means of the degenerate PCR and genomic walking technology. The Fur of Edwardsiella tarda (Etfur) shares 90% overall sequence identity with the Fur of E.coli (Ecfur). The results of functional analysis demonstrated that FurEt could recognize the target promoter of Ecfur and inhibit its activity. The results of functional domain analysis demonstrated that C92S and C95S mutations inactivated Etfur whereas E112K mutation resulted in a superactive Etfur variant. Analyzing the activities of etfur promoter showed that Etfur negatively regulated its own expression. The etfur promoter was identified by means of site-directed mutations and promoter mapping. To investigate what would ensue from disruption of the regulated synthesis of Etfur, the strain TX1/pJRTF that overexpressing etfur was created. Comparing with the wildtype TX1/pJRA, TX1/pJRTF exhibited slower doubling time and lower maximum cell density. To investigate whether the alteration in growth was accompanied by any alteration in protein production, the outer membrane proteins of TX1/pJRTF and TX1/pJRA were extracted, which showed that, comparing to TX1/pJRA, TX1/pJRTF exhibited similar but distinctly different protein profiles, suggesting that overexpression of etfur changed the production of certain outer membrane proteins. Then, the different proteins were extracted for the identification of the genes encoding these proteins by means of the reversed western immunoblotting. Virulence test indicated that overexpression of etfur attenuated bacterial virulence. We next anlaysed the promoter activity of the haemolysin gene ethB and found that Etfur could repress the expression of the ethB. Finally, we constructed a selection systerm for the screening of repressive transcription regulators. By using this system we discovered a second and more effective ethB regulator, EthR, which is a member of the GntR family of transcription regulator.
页数	109
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/999
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	王芳. 迟缓爱德华氏菌铁调控蛋白与溶血素调控蛋白的分子生物学研究[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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