Gueguen M, Yamamoto A M, Bernard O, Alvarez F
INSERM U 56, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France.
Biochem Biophys Res Commun. 1989 Mar 15;159(2):542-7. doi: 10.1016/0006-291x(89)90027-2.
Anti-liver-kidney microsome antibody type 1 (LKM1), present in the sera of a group of children with autoimmune hepatitis, was recently shown to recognize a 50 kDa protein identified as rat liver cytochromes P450 db1 and db2. High homology between these two members of the rat P450 IID subfamily and human P450 db1 suggested that anti-LKM1 antibody is directed against this human protein. To test this hypothesis, a human liver cDNA expression library in phage lambda GT-11 was screened using rat P450 db1 cDNA as a probe. Two human cDNA clones were found to be identical to human P450 db1 by restriction mapping. Immunoblot analysis using as antigen, the purified fusion protein from one of the human cDNA clones showed that only anti-LKM1 with anti-50 kDa reactivity recognized the fusion protein. This fusion protein was further used to develop an ELISA test that was shown to be specific for sera of children with this disease. These results: 1) identify the human liver antigen recognized by anti-LKM1 auto-antibodies as cytochrome P450 db1, 2) allow to speculate that mutation on the human P450 db1 gene could alter its expression in the hepatocyte and make it auto-antigenic, 3) provide a simple and specific diagnostic test for this disease.
1型抗肝肾微粒体抗体（LKM1）存在于一组自身免疫性肝炎患儿的血清中，最近研究表明，该抗体可识别一种50 kDa的蛋白质，该蛋白质被鉴定为大鼠肝细胞色素P450 db1和db2。大鼠P450 IID亚家族的这两个成员与人类P450 db1具有高度同源性，这表明抗LKM1抗体是针对这种人类蛋白质的。为了验证这一假设，以大鼠P450 db1 cDNA为探针，筛选了噬菌体λGT-11中的人类肝脏cDNA表达文库。通过限制性图谱分析发现，两个人类cDNA克隆与人类P450 db1相同。使用其中一个人类cDNA克隆的纯化融合蛋白作为抗原进行免疫印迹分析，结果显示，只有具有抗50 kDa反应性的抗LKM才能识别该融合蛋白。该融合蛋白进一步用于开发一种ELISA检测方法，结果表明该方法对患有这种疾病的患儿血清具有特异性。这些结果：1）确定抗LKM1自身抗体识别的人类肝脏抗原为细胞色素P450 db1；2）推测人类P450 db1基因的突变可能会改变其在肝细胞中的表达，使其成为自身抗原；3）为这种疾病提供了一种简单而特异的诊断检测方法。
