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Molecular analysis of MPO for patient with autoimmune disease ANCA

自身免疫性疾病 ANCA 患者 MPO 的分子分析

基本信息

批准号：
05670212
负责人：
SUZUKI Kazuo
金额：
$ 1.54万
依托单位：
National Institute of Health
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

In pauci-immune necrotizing crescentic glomerulonephritis (NCGN) autoimmune disease, anti myeloperoxidase (MPO) -antibody increase in peripheral blood of the patients.Determination of epitopes of anti-MPO antibody in some autoimmune diseases such as NCGN my give us a clue to the etiology and be used for prediction of the diseases.We have demonstrated three types of MPO (I,II,and III) and that some sera of GN reacted with the large subunit (59 kDa) of MPO-III,but not to the small subunit (14 kDa).As one serum of them most strongly reacts with a 55-kDa-deglycosylated form derived from the 59-kDa large subunit, we have attempted to prepare some recombinant the large fragments of large subunit.In the present study, we prepared the recombinant MPO-fragments as panels to analyze epitope of serum of patient with autoimune disease.cDNA encoding 59 kDa-subunit (large) of MPO was amplified by PCR in 12 parts and these cDNAs were separately inserted into the vector.The recombinant MPO fragments were expressed in E.coli and were digested with guanidine hydrochloride in a sonicator.The fragments were purified with an affinity column chromatography.The obtained fragments were used for western blot analysis to determine the reactivity of sera of patients.Some sera reacted with the MPO fragments containing Met409.The results confirmed the recognition site in native MPO-III molecule by an anti-MPO serum is around sugar attachment sites in the N-terminus in the large subunit of MPO.

在少免疫性坏死性新月体肾炎（NCGN）等自身免疫性疾病中，患者外周血中抗髓过氧化物酶（MPO）抗体升高。测定NCGN等自身免疫性疾病中抗MPO抗体的表位，可为疾病的病因诊断提供线索，并可用于疾病的预测。本文报道了NCGN等自身免疫性疾病中MPO抗体的三种类型（Ⅰ、Ⅱ、Ⅲ），部分GN患者血清与MPO-Ⅲ大亚基（59 kDa）反应，但与小亚基（14 kDa）不反应，其中一种血清与59 kDa大亚基衍生的55 kDa去糖基化形式反应最强，我们尝试制备一些重组的大亚基的大片段。在本研究中，我们制备了重组MPO片段作为分析自身免疫病患者血清表位的面板。用PCR方法扩增出MPO基因的12个片段，分别插入到载体中，在大肠杆菌中表达，用盐酸胍超声酶切，纯化后，用SDS-PAGE分析，得到目的片段。将获得的片段进行Western blot分析，部分患者血清与含Met 409的MPO片段发生反应，结果证实抗MPO血清对天然MPO-Ⅲ分子的识别位点位于MPO大亚基N-末端的糖结合位点附近。