Identification and characterization of Haemophilus parainfluenzae antigens associated with IgA nephropahty

虽然IgA肾病是日本最常见的预后不良的肾炎，但其病因尚不清楚。自从早期的报告描述IgA肾病和流感嗜血杆菌的关联，我们已经开始调查，以确定副流感嗜血杆菌的抗原。在之前的研究中，我们成功地分离了一种名为hpnA的新基因，作为引起IgA肾病的候选基因。这些研究也表明存在除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。利用从IgA肾病患者中分离的6株副流感嗜血杆菌。差速离心制备外膜组分，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批抗体，并从膜上切除仅与患者血清可见的条带。胰蛋白酶化后，将样品应用于质谱仪。通过与蛋白质数据库的比对来确定样品中的蛋白质。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端有组氨酸标记的重组OMP，并将其应用于免疫印迹分析。我们在许多IgA肾病患者中发现了针对P6的IgA抗体，这表明P6可能参与了这种疾病的发病机制。

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.