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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 high purity eti gene expression intimin (Intimin) to study its adhesion. Methods The eae gene was amplified from the whole genome of enterohemorrhagic Escherichia coli (EHEC) O157: H7. After TA cloning, eae was inserted into vector pET28a (+) and transformed into E. coli BL21 (DE3) The recombinant protein was purified by -NTA agarose column. The relative molecular mass of the target protein was detected by SDS-PAGE. The immunoreactivity was analyzed by immunoblotting. The adhesion of the recombinant protein was detected by immunofluorescence. The recombinant plasmid pET28a (+) - eae was constructed and expressed in E.coli BL21 (DE3) as inclusion body in the form of inclusion bodies, Mr 97000; Ni2 + -NTA agarose column was used to purify Intimin; Escherichia coli O157: H7 multi-antiserum detected a specific Intimin band at Mr 97000; Intimin adhered to the surface of HEp-2 cells. Conclusion Intimin, a highly purified recombinant protein, possesses certain immunoreactivity and can adhere to HEp-2 cells, which lays the foundation for further study on the interaction between Intimin protein and host receptor protein.