| 肠出血性大肠杆菌O157P:H7紧密黏附素的基因克隆、表达及功能研究 |
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| 引用本文： | 彭丽娟,周勇,杨瑜,惠长野,赵卫,万成松. 肠出血性大肠杆菌O157P:H7紧密黏附素的基因克隆、表达及功能研究[J]. 南方医科大学学报, 2009, 29(4) |
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| 作者姓名： | 彭丽娟 周勇 杨瑜 惠长野 赵卫 万成松 |
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| 作者单位： | 彭丽娟,杨瑜,惠长野,赵卫,万成松,PENG Li-juan,YANG Yu,HUI Chang-ye,ZHAO Wei,WAN Cheng-song(南方医科大学公共卫生与热带医学学院微生物学系,广东,广州,510515);周勇,ZHOU Yong(广州市疾病预防控制中心,广东,广州,510380) |
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| 摘 要： | 目的 获得高纯度的eae基因表达蛋白紧密黏附素(Intimin),研究其黏附作用.方法 从肠出血性大肠杆菌(EHEC)O157:H7全基因组中扩增出eae基因,T-A克隆后,将eae插入载体pET28a(+),并转化至E.coli BL21(DE3)中表达;用Ni2+-NTA琼脂糖柱纯化出重组蛋白;SDS-PAGE检测目的 蛋白相对分子质量,免疫印记分析其免疫反应性,免疫荧光检测其黏附性.结果 获得了大小约2805 bp的eae片段;构建r重组载体pET28a(+)-eae,并在E.coli BL21(DE3)中以包涵体形式表达Intimin,Mr约97 000;Ni2+-NTA琼脂糖柱纯化出Intimin;大肠杆菌O157:H7多抗血清在Mr约97 000处检测出一条特异性Intimin带;Intimin可黏附在HEp-2细胞表面.结论 高纯度的重组蛋白Intimin具有一定的免疫反应性,能与HEp-2细胞黏附,为进一步研究Intimin蛋白与宿主受体蛋白的相互作用奠定基础.
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| 关 键 词： | 肠出血性大肠杆菌,O157 紧密黏附素 基因克隆 重组表达 免疫印记 免疫荧光 |
Gene cloning,prokaryotic expression and functional evaluation of intimin from enterohemorrhagic Escherichia coli O157:H7 |
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| PENG Li-juan,ZHOU Yong,YANG Yu,HUI Chang-ye,ZHAO Wei,WAN Cheng-song. Gene cloning,prokaryotic expression and functional evaluation of intimin from enterohemorrhagic Escherichia coli O157:H7[J]. Journal of Southern Medical University, 2009, 29(4) |
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| Authors: | PENG Li-juan ZHOU Yong YANG Yu HUI Chang-ye ZHAO Wei WAN Cheng-song |
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| Affiliation: | PENG Li-juan1,ZHOU Yong2,YANG Yu1,HUI Chang-ye1,ZHAO Wei1,WAN Cheng-song1 1Department of Medical Microbiology,School of Public Health and Tropical Medicine,Southern Medical University,Guangzhou 510515,China,2Guangzhou Center for Disease Control and Prevention,Guangzhou 510380 |
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| Abstract: | Objective To obtain highly purified intimin encoded by the eae gene and study its adhesion activity. Methods The eae gene was amplified from enterohemorrhagic Escherichia coli O157:H7 (EHEC) chromosome by PCR and cloned into pMD19-T vector. The eae gene was cut from pMD19-T vector and subcloned into the prokaryotic expression plasmid pET28a (+), and expressed in E.coli BL21 (DE3). The recombinant protein was purified with Ni2+-chelating affinity chromatography followed by identification with SDS-PAGE and We... |
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| Keywords: | H7 eae |
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