Molecular analysis of MPO for patient with autoimmune disease ANCA

在少免疫坏死性新月体肾炎(NCGN)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。某些自身免疫性疾病(如NCGN)中抗MPO抗体表位的测定可以为我们提供病因线索，并用于疾病的预测。我们证明了MPO的三种类型(I，II和III)，一些GN血清与MPO-III的大亚基(59 KDa)反应，而不与小亚基(14 KDa)反应。作为其中最强反应的血清之一，与59 kDa大亚基衍生的55 kDa脱糖形式的GN，我们试图制备一些重组的大亚基大片段。在本研究中，为分析自身免疫病患者血清中的MPO表位，我们制备了重组MPO片段。通过聚合酶链式反应扩增出编码59 kDa亚基(大亚基)的cDNA12个片段，并将其分别插入到载体中。重组MPO片段在大肠杆菌中表达，经声学分析后，经亲和层析纯化，获得的片段用于免疫印迹分析。部分血清与含有Met409的MPO片段发生反应，结果证实了天然MPO-III分子中的识别位点。抗MPO血清位于MPO大亚基N-末端的糖结合部位附近。

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.