Molecular characterization of epitope (s) reactive with antibodies to asialoglycoprotein receptor in patients with autoimmune hepatitis

用聚合酶链反应从AGPR-H1全长cDNA克隆中获得了编码AGPR-H1胞内外结构域的部分cDNA片段。利用这些cDNA片段，在大肠杆菌中表达了胞外和胞内结构域的重组AGPR蛋白。纯化重组蛋白，用酶联免疫吸附试验（ELISA）检测自身免疫性肝炎（AIH）患者血清中的AGPR抗体。用AGPR蛋白的胞外区进行ELISA检测，AIH患者对AGPR抗体的敏感性相似，而AIH和慢性丙型肝炎患者对AGPR抗体的反应性较低。应用免疫沉淀法，使用生物素化的重组AGPR蛋白作为抗原和蛋白G-琼脂糖作为载体。在ELISA中显示高GD值的AIH血清显示与细胞外AGPR蛋白的强免疫沉淀，并且没有表达AGPR蛋白。 ...更多信息 与蛋白质的胞内结构域显著沉淀。在ELISA和免疫沉淀中仅检测到弱阳性信号，表明AIH中与AGPR抗体反应的表位可能在SDS丙烯酰胺凝胶电泳中被降解。ELISA和免疫沉淀结果表明，与AIH抗体反应的主要抗原表位可能位于AGPR蛋白的胞外区。这一发现支持抗AGPR抗体可能在AIH中起致病作用。肝细胞损伤可通过抗体依赖性细胞介导的细胞毒作用产生。用重组AGPR抗原进行的淋巴细胞刺激试验显示，几例AIH患者外周血单个核细胞中的淋巴细胞有明显的增殖反应。进一步鉴定AGPR抗原上的B和T细胞表位对于阐明AIH中对AGPR的自身免疫应答的病理学意义是必要的。少

The partial cDNA fragments encoding the extra- and intracellular domain of asialoglycoprotein receptor (AGPR)-Hl was obtained by polymerase chain reaction from a full-length AGPR-Hl cDNA clone. Using these cDNA fragments, recombinant AGPR proteins of extra- and intracellular domain were expressed in E.coli. Recombinant proteins were purified and subjected to an enzyme-linked immunosorbent assay (ELISA) to detect antibodies to AGPR in sera from autoimmune hepatitis (AIH), By using extracellular domain of AGPR protein in ELISA, a similar sensitivity to pick-up anti-AGPR antibodies was demonstrated in AIH and less reactivity was detected in AIH and chronic hepatitis C.In order to confirm an antigen-antibody system in AIH, an immunoprecipitation method was applied using biotinilated recombinant AGPR proteins as antigen and Protein G-agarose as carrier. Sera from AIH, which showed high GD values in ELISA, demonstrated a strong immunoprecipitation with the extracellular AGPR protein and no s … More ignificant precipitation with intracellular domain of the protein. Weakly positive signals were only detected with sera which gave a strong reactivity in ELISA and immunoprecipitation, suggesting that epitope(s) reactive with antibodies to AGPR in AIH might be degraded during SDS acrylamide gel electrophoresis. The results from ELISA and immunoprecipitation suggested that main epitope(s), which were reactive with antibodies in AIH, could be located on the extracellular domain of the AGPR protein. This finding support that anti-AGPR antibodies might play an pathogenic role in AIH.Hepatocytic injury could be generated via antibody-dependent cell-mediated cytotoxicity. Lymphocytes from peripheral blood mononuclear cells in several AIH patients showed a significant proliferation in a lymphocyte stimulation test with recombinant AGPR antigen. Further characterization of B and T cell epitope(s) on AGPR antigens is necessary to elucidate the pathological significance of autoimmune response to AGPR in AIH. Less