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Identification and characterization of Haemophilus parainfluenzae antigens associated with IgA nephropahty

与 IgA 肾病相关的副流感嗜血杆菌抗原的鉴定和表征

基本信息

批准号：
15590384
负责人：
TRII Keizo
金额：
$ 1.98万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590384/
关键词：
IgA nephropathy Haemophilus parainfluenzae mass spectrometer IgA腎 H.parainfluenzae

项目摘要

Although IgA nephropathy is the most frequent nephritis that has poor prognosis in Japan, etiology of this disease is still unknown. Since the early report describing the association of IgA nephropathy and Haemophilus influenzae, we have started investigation to identify the antigen (s) of H.parainfluenzae. In previous studies, we successfully isolated the novel gene, named hpnA, as a candidate that cause IgA nephropathy. Those studies also indicated the existence of antgens other than hpnA. Therefore, we designed current experiments to find those antigens. Six strains of H.parainfluenzae isolated irom patients with IgA nephropathy were utilized. Outer membrane fractions were prepared by differential centrifugation, and separated by SDS-PAGE. For immunoblot analysis, sera from patients or healthy volunteers were used for the first antibodies, and bands only visible with patients' sera were excised from membrane. After trypsinization, samples were applied onto mass spectrometer. Proteins in samples were determined by comparison towards protein database. Results of these experiments indicated that OMP and P6 were newly identified In H.parainfluenzae, nucleotide sequence for P6 has been determined, we synthesized recombinant OMP that has histidine-tag at N-terminus, and applied it to immunoblot analysis. We found the existence of IgA antibodies against P6 in many patients with IgA nephropathy, indicating the possible involvement of P6 in the pathogenesis of this disorder.

尽管IgA肾病是日本最常见且预后不良的肾炎，但该病的病因仍不清楚。自从早期报道描述 IgA 肾病与流感嗜血杆菌之间的关联以来，我们已开始研究鉴定副流感嗜血杆菌的抗原。在之前的研究中，我们成功分离出一种新基因，命名为hpnA，作为导致IgA肾病的候选基因。这些研究还表明存在 hpnA 以外的抗原。因此，我们设计了当前的实验来寻找这些抗原。使用从患有 IgA 肾病的患者中分离出的六种副流感嗜血杆菌菌株。通过差速离心制备外膜级分，并通过 SDS-PAGE 分离。对于免疫印迹分析，使用患者或健康志愿者的血清作为第一抗体，并从膜上切除仅在患者血清中可见的条带。胰蛋白酶消化后，将样品应用到质谱仪上。通过与蛋白质数据库的比较来确定样品中的蛋白质。实验结果表明，在副流感嗜血杆菌中，OMP和P6是新鉴定的，P6的核苷酸序列已确定，我们合成了N端带有组氨酸标签的重组OMP，并将其应用于免疫印迹分析。我们发现许多 IgA 肾病患者体内存在针对 P6 的 IgA 抗体，表明 P6 可能参与该疾病的发病机制。