致病微生物与抗肾小球基底膜病发病机制研究

Anti-glomerular basement membrane (GBM) disease is the most severe glomerulonephritis without a known etiology. Due to flu-like prodromal symptoms in most patients, we hypothesized that anti-GBM disease is induced by microbes, through molecular mimicry theory, by cross-reaction between microbial proteins and human α3(IV)NC1. Identification of the key amino acid motif in epitopes on α3(IV)NC1 is crucial to detect the possible microbes. Our previous work demonstrated a possible initial linear epitope P14c on α3(IV)NC1 molecule as mutual T and B cell epitope with an key amino acid motif GFxF. Using NCBI protein Blast tool, we identify a polysaccharide biosynthesis protein of Bacteroidetes oral taxon, which shows a high amino acid sequence homology with P14c and contains the GFxF motif. We further hypothesized that Bacteroidetes oral taxon might be the causal microbe of anti-GBM disease, but without commercial available resource. The current study aims: (1) to produce recombinant polysaccharide biosynthesis protein of Bacteroidetes oral taxon or to synthesize antigenic peptides based on predicted epitopes and GFxF motif on the microbial protein, the recombinant protein or antigenic peptides will be used as antigens to detect infection rate of patients with anti-GBM disease; (2) to immunize anti-GBM disease susceptible Wistar Kyoto rats with the above recombinant protein and antigenic peptides to induce rat anti-GBM nephritis. This study will not only clarify the possible etiology of anti-GBM disease and pathogenesis of switching from microbial antigen to auto antigen, but also will provide useful biomarkers and potential therapeutic targets.

抗肾小球基底膜(GBM)病是最严重的肾炎，病因不清。发病前多有前驱感染症状，推测病原微生物与人GBM抗原α3(IV)NC1存在交叉反应，通过分子模拟机制诱发疾病。确定自身抗原的初始线性抗原决定簇是寻找致病微生物的关键。前期研究发现α3(IV)NC1的线性肽P14c是T/B细胞共有的抗原决定簇并确定了核心氨基酸基序GFxF，利用NCBI蛋白质Blast工具找到的口腔拟杆菌多糖合成蛋白可被患者血清识别，推测该细菌是抗GBM病的病因之一。但目前无法获得该菌株，本课题拟：(1)利用蛋白质重组技术表达口腔拟杆菌多糖合成蛋白，制备含有该蛋白的抗原决定簇和含有GFxF基序的抗原肽，检测患者血清抗体明确感染情况；(2)应用细菌蛋白及抗原肽免疫易感抗GBM病的WKY大鼠诱发抗GBM肾炎。本研究可明确抗GBM病的病因，阐明从细菌抗原到自身抗原的扩展机制。

抗肾小球基底膜(glomerular basement membrane, GBM)病是经典的自身免疫性肾脏病，预后差、病因不清，但起病时多伴有前驱感染，故推测病原微生物可能与人GBM存在交叉反应，通过分子模拟机制诱发疾病。我们的前期工作在肾小球基底膜α3链上发现了一个T/B细胞共有的抗原表位P14，是启动抗GBM病的初始位点，该决定簇是寻找致病微生物的关键。我们利用氨基酸替换技术，明确了P14的关键基序，并据此在NCBI、Uniprot蛋白质数据库中对微生物进行了检索，进而通过T/B细胞抗原预测软件筛选出36条微生物肽段。研究证实, 抗GBM病患者确实存在识别上述微生物肽段的自身抗体，且与肾脏损伤相关。研究还发现，来自放线菌的肽段B7可以诱发大鼠的抗GBM肾炎，该肽段与P14存在T细胞及B细胞水平上的交叉反应，并且能够结合抗GBM病的易感HLA分子DRB1*1501。在HLA-DRB1*1501转基因小鼠中，利用B7免疫动物再次诱发了抗GBM肾炎，从而为分子模拟机制参与抗GBM病的发病提供了证据。