Detection of anti-type 3 muscarinic acetylcholine receptor Autoantibodies in the sera of Sjogren's Syndrome patients by use of a transfected cell line assay
Detection of anti-type 3 muscarinic acetylcholine receptor Autoantibodies in the sera of Sjogren's Syndrome patients by use of a transfected cell line assay
复制标题
DOI：
10.1002/art.20371
复制
发表时间：
2004-08-01
影响因子：
--
通讯作者：
Peck, AB
中科院分区：
文献类型：
--
作者：
Gao, JH;Cha, SH;Peck, AB
Objective.干燥综合征（SS）是一种自身免疫性疾病，主要影响唾液腺和泪腺，导致口干和眼干。最近的研究表明，与唾液腺和泪腺细胞上表达的3型毒蕈碱乙酰胆碱受体（M3 R）反应的自身抗体可能对SS具有高度特异性。为了验证这一假设，我们构建了一个表达人M3 R基因的细胞系，以筛选SS患者血清中的抗M3 R自身抗体。扩增编码人M3 R基因的开放阅读框（ORF）的互补DNA，连接到pcDNA 5/FRT/V5-His-TOPO TA载体中，然后用于转化大肠杆菌细菌。使用Flp重组酶介导的位点特异性重组，将含有具有正确方向的M3 R ORF的质粒DNA转染到Flp-In中国仓鼠卵巢（CHO）细胞中。用潮霉素筛选并增殖M3 R基因转染的CHO细胞系，用流式细胞术检测SS患者和健康对照血清中抗M3 R自身抗体。检测血清中是否存在与CHO转染细胞结合的抗M3 R自身抗体，结果显示SS患者中存在抗M3 R自身抗体（9/11），而健康对照中不存在（0/11）。虽然在患者血清中检测到的抗M3 R自身抗体具有多种同种型，但最一致的检测是IgG 1、IgG 3和IgA。使用新构建的表达人M3 R的细胞系，抗M3 R自身抗体很容易检测到SS患者血清中。这些自身抗体倾向于IgG 1、IgG 3和伊加同种型，可能识别由受体蛋白的胞外结构域产生的三级表位。抗M3 R自身抗体代表了用于鉴定SS的非常有前途的临床标志物。
Objective. Sjogren's syndrome (SS) is an autoimmune disease affecting primarily the salivary and lacrimal glands, leading to dry mouth and dry eyes. Recent studies have suggested that autoantibodies reactive with the type 3 muscarinic acetylcholine receptors (M3Rs) expressed on salivary and lacrimal gland cells may be highly specific for SS. To test this hypothesis, we constructed a cell line expressing the human M3R gene in order to screen for anti-M3R autoantibodies in sera from SS patients.Methods. Complementary DNA encoding the open-reading frame (ORF) of the human M3R gene was amplified, ligated into the pcDNA5/FRT/V5-His-TOPO TA vector, and then used to transform Escherichia coli bacteria. Plasmid DNA containing the M3R ORF with the correct orientation was transfected into Flp-In Chinese hamster ovary (CHO) cells using Flp recombinase-mediated site-specific recombination. An M3R-transfected CHO cell line, selected and propagated in hygromycin, was used to detect anti-M3R autoantibodies in SS patient and healthy control sera by flow cytometry.Results. Testing of sera for the presence of anti-M3R antoantibodies bound to CHO-transfected cells revealed the presence of anti-M3R autoantibodies in SS patients (9 of 11) but not in healthy controls (0 of 11). Although the anti-M3R autoantibodies detected in patient sera were of multiple isotypes, the most consistently detected were IgG1, IgG3, and IgA.Conclusion. Using a newly constructed cell line expressing human M3R, anti-M3R autoantibodies were easily detected in sera from SS patients. These autoantibodies were skewed toward the IgG1, IgG3, and IgA isotypes, probably recognizing a tertiary epitope created by extracellular domains of the receptor protein. Anti-M3R autoantibodies represent a highly promising clinical marker for the identification of SS.