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

日本立克次体是我们在东方动物区分离鉴定出的一种特殊的斑点热群立克次体病，是矿源性东方斑点热病的病原。日本血吸虫的主要外膜蛋白rompA和rompB具有免疫原性，在进入宿主细胞和防御感染中发挥重要作用。本研究根据ompA和ompB基因的序列设计引物，将ompA和ompB基因的PCR产物插入表达载体pET-21a-d(+)，转化大肠杆菌BL2I(DE3)(登录号：D28766，AB003681)。用IPTG将重组蛋白表达为包涵体，用尿素溶解，然后用亲和柱层析进行纯化。经SDS-聚丙烯酰胺凝胶电泳法和Western免疫印迹法检测均为单一条带。然后在伴侣蛋白Groe和ATP存在的情况下透析，以重折叠以显示可溶的性质。这些重组蛋白和抗ROMP A和ROMP B的单特异性抗体被用来检测它们对立克次体附着在性欲细胞上的影响。用单特异性抗体和重组蛋白都能在一定程度上抑制黏附，但不能完全抑制黏附。此外，由于重组蛋白的构象与生物体上蛋白质的构象不同，因此构象的重要性被提出。从口腔鳞癌患者中分离的9株致病性立克次体也以romP A和roMP B为标记蛋白进行了表型和基因分型。结果表明，这些菌株属于单一类型，其表面主要抗原蛋白romP A和romP B可作为SFG立克次体分型的标记。

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.