Isolation and characterization of cDNA encoding a human nuclear antigen predominantly recognized by autoantibodies from patients with primary biliary cirrhosis.
Isolation and characterization of cDNA encoding a human nuclear antigen predominantly recognized by autoantibodies from patients with primary biliary cirrhosis.
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DOI：
10.4049/jimmunol.145.12.4338
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发表时间：
1990-12
影响因子：
4.4
通讯作者：
C. Szostecki;H. Guldner;H. Netter;H. Will
中科院分区：
文献类型：
--
作者：
C. Szostecki;H. Guldner;H. Netter;H. Will
一种新型的核抗原Sp100的自身抗体最近被描述为识别一种表观分子质量为95-100 kDa的核蛋白，并在细胞核内呈点状分布。用抗Sp100自身免疫血清对HeLa细胞中的lambda gt11基因表达文库进行免疫筛选，获得了编码Sp100基因片段的0.7kb的cDNA(Sp26)。该基因的表达和重组蛋白在ELISA中的应用表明，该片段携带主要的Sp100自身表位，抗Sp100自身抗体主要发生在原发性胆汁性肝硬变患者(50/184)。以Sp26作为杂交探针，从人肝脏和胎盘来源的lambda gt10文库中分离出较长的cDNA.将重叠片段组装成编码一个分子质量为53 kDa、等电点为4.7的蛋白质的全长cDNA。Sp100自身抗原在体外表达，并经捕获免疫印迹试验鉴定，在SDS-PAGE中与95~100 kDa的HeLa自身抗原融合，表现出异常的电泳率。基于计算机的Sp100自身抗原的蛋白质序列分析显示，与各种人和非人MHC I类抗原的α1和α2结构域以及几种转录调节蛋白具有显著的序列相似性。
Autoantibodies to a novel nuclear Ag, Sp100, have recently been described that recognize a nuclear protein with an apparent molecular mass of 95 to 100 kDa and a dot-like distribution within cell nuclei. By immunoscreening of a lambda gt11 cDNA expression library derived from HeLa cells with an anti-Sp100 autoimmune serum a 0.7-kb cDNA (Sp26) coding for a fragment of Sp100 was isolated. Expression of this cDNA and use of the recombinant protein in ELISA revealed that the fragment carries major Sp100 autoepitopes and that anti-Sp100 autoantibodies predominantly occur in patients suffering from primary biliary cirrhosis (50/184). The Sp26 cDNA was used as hybridization probe for isolation of longer cDNA from human liver- and placenta-derived lambda gt10 cDNA libraries. Overlapping fragments were assembled to generate a full length cDNA coding for a protein with a molecular mass of 53 kDa and an isoelectric point of 4.7. The Sp100 autoantigen expressed in vitro from this cDNA and authenticated by a capture immunoblot assay, comigrated in SDS-PAGE with the authentic HeLa autoantigen of 95 to 100 kDa and thus showed an aberrant electrophoretic mobility. Computer based protein sequence analysis of the Sp100 autoantigen revealed regions of striking sequence similarities to the alpha 1 and alpha 2 domains of various human and non-human MHC class I Ag and to several transacting transcriptional regulatory proteins.