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  

Gene cloning,prokaryotic expression and functional evaluation of intimin from enterohemorrhagic Escherichia coli O157:H7

作　　者：彭丽娟[1] 周勇[2] 杨瑜[1] 惠长野[1] 赵卫[1] 万成松[1] 

机构地区：[1]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 [2]广州市疾病预防控制中心,广东广州510380

出　　处：《南方医科大学学报》2009年第4期707-710,共4页Journal of Southern Medical University

基　　金：广东省NBC项目(2006B33761001-13)

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2805bp的eae片段;构建了重组载体pET28a(+)-eae,并在E.coli BL21(DE3)中以包涵体形式表达Intimin,Mr约97000;Ni2+-NTA琼脂糖柱纯化出Intimin;大肠杆菌O157:H7多抗血清在Mr约97000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。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 eoe 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 Ni^2＋-chelating affinity chromatography followed by identification with SDS-PAGE and Western blotting. The purified intimin was detected by immunofluorescence staining to test its adhesion. Results The 2805-bp eae gone fragment was obtained, and the recombinant expression plasmid pET28a （＋）-eoe was successfully expressed in E.coli BL21 （DE3）. The molecular weight of the recombinant protein was 97 000. Purified recombinant intimin was recognized by rabbit anti-O157 antiserum, and bound to the surface of HEp-2 cells as revealed by immunofluorescence staining. Conclusion Highly purified and immunoreactive intimin has been successfully obtained, which can adhere to the surface of HEp-2 cells. The acquisition of recombinant intimin provides the basis for studying its interaction with the host receptors during EHEC O 157：H7 infection.

关 键 词：肠出血性大肠杆菌 0157:H7 EAE 紧密黏附素 基因克隆 重组表达 免疫印记 免疫荧光 

分 类 号：R378.21[医药卫生—病原生物学]