自身免疫性肝炎特异性靶抗原的表达及免疫学鉴定

Expression and identification of specific autoantigens in autoimmune hepatitis

目的 克隆、表达可溶性肝抗原（ＳＬＡ）及细胞色素Ｐ４５０ ２Ｄ６（ＣＹＰ ２Ｄ６）。方法 采用 ＲＴ—ＰＣＲ技术从人肝组织ｐｏｌｙ（Ａ）＋ＲＮＡ中扩增ＳＬＡ及 ＣＹＰ ２Ｄ６ ｃＤＮＡ，经ＢａｍＨ Ⅰ、Ｈｉｎｄ Ⅲ双酶切定向插入载体ＰＱＥ－３０并在大肠杆菌Ｍ１５中表达。对表达载体ＰＱＥ—３０／ＳＬＡ、ＰＱＥ—３０／ＣＹＰ ２Ｄ６中的目的基因进行序列分析，表达产物用 ＳＤＳ—ＰＡＧＥ、免疫印迹方法鉴定。结果 表达产物经 ＳＤＳ－ＰＡＣＥ和免疫印迹分析后在分子量 ４．７ × １０４和５．０ × １０４处各有一条明显的蛋白带，并分别能特异性与抗ＳＬＡ、ＣＹＰ２Ｄ６阳性血清反应。结论 表达ＳＬＡ、ＣＹＰ２Ｄ６为自身免疫性肝炎的诊断及其发病机制研究提供物质基础。

Objective To express and identify soluble liver antigen (SLA) and cytochrome P-450 (CYP 2D6). Methods SLA cDNA and CYP 2D6 cDNA were obtained from human liver tissue poly (A)+RNA by RT-PCR. The cDNAs were inserted into fusion expression vector PQE-30 site of BamH I and Hind III. SLA and CYP 2D6 were identified by the SDS-PAGE and Western blot. Results SDS-PAGE analysis showed that there was a very strong stained band at about 47 kd and 50 kd, respectively. The products could specifically band to anti-SLA or anti-CYP 2D6 autoantibodies. Conclusions The clone and expression of SLA and CYP 2D6 provide usetlil substances for the diagnosis and research of pathogenesis on autoimmune hepatitis.