去唾液酸糖蛋白受体H1亚单位表达及用于自身免疫性肝炎诊断的研究

Prokaryotic expression of asialoglycoprotein receptor H1 subunit and its application in the diagnosis of autoimmune hepatitis

去唾液酸糖蛋白受体（asialoglycoprotein receptor,ASGPR）是肝细胞膜表面特有的一种内吞性受体,H1是参与内吞功能的主要亚基。自身免疫性肝炎（autoimmune-hepatitis,AIH）患者存在抗-ASGPR的自身抗体。本研究以去唾液酸糖蛋白受体H1亚基在大肠杆菌中诱导表达,用纯化的重组去唾液酸糖蛋白受体H1亚单位rH1作为检测抗原,建立间接法rH1-IgG-ELISA,检测抗去唾液酸糖蛋白受体IgG抗体,观察其阳性和阴性符合率,并对rH1-IgG-ELISA检测的精确度、灵敏度和特异性进行评价。结果显示制备的rH1重组蛋白纯度在90%以上;以该重组抗原建立的rH1-IgG-ELISA的最佳检测条件为：重组抗原rH1的包被浓度为6μg/ml,血清稀释度1：100,酶标记的羊抗人IgG 1：3000稀释;用rH1-IgGELISA对混合ASGPR阳性和阴性血清的重复检测表明：IgG阳性血清的检测值的变异系数（CV值）为9.9%,IgG阴性血清的CV值为9.7%;灵敏度检测表明血清稀释度在1：50~1：200均可检出阳性;特异性试验的抑制率为63.5%;rH1-IgG-ELISA与总符合率为87.36%,其中阳性和阴性符合率分别为82%（41/50）和93.33%（42/45）。故建立的rH1-IgGELISA具有较好的灵敏性和特异性,与提供的ASGPR阳性血清有较高的符合率,表明该法具有较好的AIH诊断价值,可进一步推广应用。

Asialoglyeoprotein receptor（ASGPR）is an internalizing receptor on hepatic cells,and its H1 subunit is importantly involved in the process of internalization.There are anti-ASGPR autoantibodies in patients with autoimmune-hepatitis（AIH）.This study aims to establish an immune enzyme assay for diagnosis of HIA by using recombinant receptor subunit H1 expressed in prokaryotic system to detect autoantibodies against ASGPR in human sera.The sensitivity,specificity,negative rate and positive rate were also assessed.The results showed that the recombinant H1 protein of molecular mass weight about 50.3 kD was expressed in E.coli as inclusion body;rHl was prepared with Ni^2＋column purification;A polyclonal antibody was also successfully prepared by immunization with H1 recombinant protein and was proved by Western blot analysis of 6his-H1 and GST-H1 recombinant protein.High-level expression of H1 was achieved in E.coli in this study,and H1 expression can now be monitored in vitro and in vivo gene transfer studies with the polyclonal antibody.The polyclonal antibody can be used to develop a new enzyme-linked immunosorbent assays（ELISA）for quantification of the soluble asialoglyeoprotein receptor.