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.

我们将japonica的立克人分离并被我们鉴定，这是该疾病开采的东方斑点发烧（OSF）的病因，作为东方动物区域中特定的斑点发烧组（SFG）的立克氏病。众所周知，主要的外膜蛋白会在进入宿主细胞和防御感染中具有免疫原性和重要作用。在这项研究中，Escherichia coli Bl2i（De3）通过表达载体PET-21A-D（+）转化，使用基于这些基因的序列（DDBJ登录编号； D28766，AB003681），使用基于这些基因的序列的引物插入了OMPA和OMPB的PCR产物。重组蛋白表示为带有IPTG的包含体的重组蛋白用尿素溶解，然后通过亲和色谱柱色谱法进行纯化。在SDS-聚丙烯酰胺凝胶电泳和西部免疫印迹中获得的单条带。然后将蛋白质透析在伴侣蛋白浓度和ATP的情况下进行透析，以表明可溶性。检查了针对ROMP A和ROMP B的这些重组蛋白和单特异性抗体的作用，其作用是Rickettsiae附着在欲望细胞上。通过使用单特异性抗体和重组蛋白，可以观察到一定程度但未完成固定的抑制作用。此外，由于证明重组蛋白的构象与生物体上的蛋白质不同，因此提出了构象的重要性。还通过使用ROMP A和ROMP B作为标记蛋白分析了从OSF患者中分离出的9种病原Rickettsiae菌株。结果表明，这些菌株属于一种类型，并且在附件上起作用的主要表面抗原蛋白rompA和romp b可作为键入SFG Rickettsiae的标记。