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利用噬菌体展示技术对原发性胆汁性肝硬化和自身免疫性胆管炎中抗体表达的比较研究。

A comparative study of antibody expressions in primary biliary cirrhosis and autoimmune cholangitis using phage display.

作者信息

Ikuno N, Scealy M, Davies J M, Whittingham S F, Omagari K, Mackay I R, Rowley M J

机构信息

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

出版信息

Hepatology. 2001 Sep;34(3):478-86. doi: 10.1053/jhep.2001.27013.

Abstract

Primary biliary cirrhosis (PBC) and autoimmune cholangitis (AIC) are serologic expressions of an autoimmune liver disease affecting biliary ductular cells. Previously we screened a phage-displayed random peptide library with polyclonal IgG from 2 Australian patients with PBC and derived peptides that identified a single conformational (discontinuous) epitope in the inner lipoyl domain of the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the characteristic autoantigen in PBC. Here we have used phage display to investigate the reactivity of PBC sera from 2 ethnically and geographically distinct populations, Japanese and Australian, and the 2 serologic expressions, PBC and AIC. Random 7-mer and 12-mer peptide libraries were biopanned with IgG from 3 Japanese patients with PBC and 3 with AIC who did not have anti-PDC-E2. The phage clones (phagotopes) obtained were tested by capture enzyme-linked immunosorbent assay (ELISA) for reactivity with affinity-purified anti-PDC-E2, and compared with those obtained from Australian patients with PBC. Peptide sequences of the derived phagotopes and sequences derived by biopanning with irrelevant antisera were aligned to develop a guide tree based on physicochemical similarity. Both Australian and Japanese PBC-derived phagotopes were distributed in branches of the guide tree that contained the peptide sequences MH and FV previously identified as part of an immunodominant conformational epitope of PDC-E2, indicating that epitope selection was not influenced by the racial origin of the PBC sera. Biopanning with either PBC or AIC-derived IgG yielded phagotopes that reacted with anti-PDC-E2 by capture ELISA, further establishing that there is a similar autoimmune targeting in PBC and AIC.

摘要

原发性胆汁性肝硬化(PBC)和自身免疫性胆管炎(AIC)是一种影响胆管细胞的自身免疫性肝病的血清学表现。此前,我们用来自2例澳大利亚PBC患者的多克隆IgG筛选了一个噬菌体展示随机肽库,并获得了一些肽段,这些肽段在丙酮酸脱氢酶复合体(PDC-E2)E2亚基的内部硫辛酰结构域中鉴定出一个单一的构象(不连续)表位,这是PBC中的特征性自身抗原。在此,我们利用噬菌体展示技术研究了来自两个种族和地域不同人群(日本人和澳大利亚人)的PBC血清以及两种血清学表现形式(PBC和AIC)的反应性。用3例日本PBC患者和3例无抗PDC-E2的AIC患者的IgG对随机7肽和12肽文库进行生物淘选。通过捕获酶联免疫吸附测定(ELISA)检测获得的噬菌体克隆(噬菌体表位)与亲和纯化的抗PDC-E2的反应性,并与从澳大利亚PBC患者获得的噬菌体克隆进行比较。将所得噬菌体表位的肽序列与用无关抗血清进行生物淘选得到的序列进行比对,以基于物理化学相似性构建一个指导树。来自澳大利亚和日本PBC的噬菌体表位均分布在指导树的分支中,这些分支包含先前被鉴定为PDC-E2免疫显性构象表位一部分的肽序列MH和FV,这表明表位选择不受PBC血清种族来源的影响。用PBC或AIC来源的IgG进行生物淘选均产生了通过捕获ELISA与抗PDC-E2反应的噬菌体表位,进一步证实PBC和AIC存在相似的自身免疫靶向作用。

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