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Analysis of the functional domains on the major surface antigen proteins of Rickettsia japonica

日本立克次体主要表面抗原蛋白功能域分析

基本信息

批准号：
09670285
负责人：
UCHIYAMA Tsuneo
金额：
$ 1.92万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670285/
关键词：
Rickettsia japonica Oriental spotted fever the major surface antigen rOmp A rOmp B adherence recombinant protein invasion Rickettsia Japonica 機能ドメイン

项目摘要

Rickettsia japonica was isolated and identified by us which is the causative agent of the disease mined Oriental spotted fever (OSF) as a specific spotted fever group (SFG) rickettsiosis in the Oriental faunal region. The major outer membrane proteins rOmp A and rOmp B of R.japonica are known to possess immunogenicity and important roles in the entry into the host cells and in the defense from infection. In this research, Escherichia coli BL2I(DE3) was transformed by expression vector pET-21a-d(+) which was inserted by PCR products of ompA and ompB using primers designed based m the sequences of these genes (DDBJ Accession Numbers ; D28766, AB003681). Recombinant proteins expressed as inclusion bodies with IPTG was solubilized with urea, followed by purification by affinity column chromatography. Single bands w ere obtained in SDS-polyacrylamide gel electrophoresis and Western immunoblotting. The proteins were then dialyzed in the presence of chaperonin GroE and ATP to be refolded to show the soluble nature. These recombinant proteins and monospecific antibodies against rOmp A and rOmp B were examined for their effects m the attachment of rickettsiae to the lust cells. Inhibition of attachment to some extent but not complete was observed by using both the monospecific antibodies and recombinant proteins. Moreover, the importance of the conformations was suggested since the conformations of the recombinant proteins are shown to be different from those of proteins on the organisms. Nine strains of pathogenic rickettsiae isolated from OSF patients were also analyzed phenotypically and genotypically using the rOmp A and rOmp B as marker proteins. It is shown that these strains belong to a single type and that the major surface antigen proteins rOmp A and rOmp B which function on the attachment are useful as markers for typing of SFG rickettsiae.

我们分离并鉴定出日本立克次体，它是东方斑疹热（OSF）疾病的病原体，是东方动物区的特定斑疹热群（SFG）立克次体病。已知R.japonica的主要外膜蛋白rOmp A和rOmp B具有免疫原性，并且在进入宿主细胞和防御感染方面发挥重要作用。在本研究中，用表达载体pET-21a-d(+)转化大肠杆菌BL2I(DE3)，该表达载体通过ompA和ompB的PCR产物插入，使用根据这些基因的序列设计的引物(DDBJ登录号；D28766，AB003681)。用 IPTG 表达为包涵体的重组蛋白用尿素溶解，然后通过亲和柱层析纯化。在 SDS 聚丙烯酰胺凝胶电泳和蛋白质免疫印迹中获得单条带。然后在伴侣蛋白 GroE 和 ATP 存在下对蛋白质进行透析，重新折叠以显示可溶性。检查了这些重组蛋白和针对 rOmp A 和 rOmp B 的单特异性抗体对立克次体附着到欲望细胞的影响。通过使用单特异性抗体和重组蛋白观察到一定程度但不完全的附着抑制。此外，由于重组蛋白的构象与生物体上蛋白质的构象不同，因此表明了构象的重要性。还使用 rOmp A 和 rOmp B 作为标记蛋白对从 OSF 患者中分离出的 9 株致病性立克次体进行了表型和基因型分析。结果表明，这些菌株属于单一类型，并且在附着物上起作用的主要表面抗原蛋白rOmp A和rOmp B可用作SFG立克次体分型的标记。