Adherence to and Invasion of Vero Cells by Recombinant Escherichia coli Expressing the Outer Membrane Protein rOmpB of Rickettsia japonica
Adherence to and Invasion of Vero Cells by Recombinant Escherichia coli Expressing the Outer Membrane Protein rOmpB of Rickettsia japonica
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表达日本立克次体外膜蛋白rOmpB的重组大肠杆菌对Vero细胞的粘附和侵袭
DOI：
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发表时间：
2003
影响因子：
5.2
通讯作者：
T. Uchiyama
中科院分区：
文献类型：
--
作者：
T. Uchiyama
摘要: 制备了表面表达立克次体外膜蛋白rOmpB的重组大肠杆菌。通过聚合酶链式反应扩增对应于斑点热群立克次体、日本立克次体的ompB基因的开放阅读框的DNA。将扩增片段插入表达载体pET-22b(+)的Sal I和Xho I位点之间。将构建的质粒转化大肠杆菌BL21(DE3)。重组细菌在表面表达了分子大小为165千道尔顿的重组蛋白。大小与rOmpB前体的大小一致。该蛋白与 rOmpB 不耐热表位的单克隆抗体发生反应。该结果表明重组蛋白具有相当天然的构象。重组细菌的免疫荧光显示了蛋白质的表面表达。重组细菌获得了进入 Vero 细胞的特性。通过透射电子显微镜和扫描电子显微镜观察形态变化。粘附触发细胞产生丰富的微绒毛和膜皱褶以吞噬细菌。重组细菌的进入方式与立克次体相似。由此表明rOmpB在立克次体对宿主细胞的粘附和侵袭中起重要作用。此外，由于即使在细菌表面表达的重组rOmpB前体蛋白也能促进粘附和侵袭，因此功能域的构象可能与加工的成熟rOmpB的构象相似。
Abstract: Recombinant Escherichia coli expressing the outer membrane protein rOmpB of rickettsiae on the surface were generated. The DNA corresponding to the open reading frame of the ompB gene of a spotted fever group rickettsia, Rickettsia japonica, was amplified by polymerase chain reaction. The amplified fragment was inserted between the Sal I and the Xho I sites of the expression vector pET‐22b(+). E. coli BL21(DE3) was transformed by the constructed plasmid. The recombinant bacteria expressed a recombinant protein with a molecular size of 165 kilodaltons on the surface. The size was consistent with that of the precursor of rOmpB. The protein was reactive with monoclonal antibodies to heat‐labile epitopes of the rOmpB. This result suggested a rather native conformation of the recombinant protein. Immunofluorescence of the recombinant bacteria demonstrated the surface expression of the protein. The recombinant bacteria acquired properties to enter Vero cells. The morphological change was observed by means of transmission electron microscopy and scanning electron microscopy. Adherence triggered the generation of abundant microvilli and membrane ruffling for the cells to engulf the bacteria The manner of entry of the recombinant bacteria was similar to that of rickettsiae. Thus it is suggested that the rOmpB plays an important role in the adherence to and invasion of host cells by rickettsiae. Moreover, since even the recombinant rOmpB precursor protein expressed on the surface of the bacteria promotes adherence and invasion, the conformation of the functional domain may be similar to that of the processed mature rOmpB.
在超薄切片上展示日本立克次体的热不稳定和热稳定表位。
DOI：
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发表时间：
1994
期刊：
Laboratory investigation; a journal of technical methods and pathology
影响因子：
--
作者：
Uchiyama,T;Uchida,T;Wen,JW;Walker,DH
通讯作者：
Walker,DH
影响因子：
3.8
作者：
Li, H;Walker, DH
通讯作者：
Walker, DH