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目的构建一株具有致病性的人抗乙酰胆碱受体(AchR)自身抗体,用于诱导猴实验性自身免疫性重症肌无力(EAMG)模型。方法将分离自重症肌无力(MG)患者胸腺的抗AchR抗体的抗原结合片段(Fab637)克隆至含有免疫球蛋白(IgG1)基因的载体pIgG1,构建能在哺乳类细胞表达完整IgG1类抗AchR抗体(IgG1-637)的重组表达载体(pIgG1-637),将pIgG1-637转染CHO-k1细胞系,筛选出稳定表达IgG1-637的细胞株扩大培养。将纯化的IgG1-637注射5只恒河猴,通过检查猴肌肉收缩强度和复合肌肉运动电位(CMAP),判定猴EAMG发病情况,从而鉴定IgG1-637的致病性。结果20μmol／L MSX(L-methionine sulfoxmine)选择的CHO-k1细胞表达最高水平的抗体IgG1-637,表达产量为17．4μg/ml。3只猴(分别接受1．7、1．7和5．0mg／kg的注射剂量)在注射IgG1-637后1周内出现肌肉收缩无力临床症状,并表现出CMAP减弱现象。结论由Fab637构建的完整的IgG1-637是一株致病性抗AchR抗体,可在猴诱导出EAMG。
Objective To construct a pathogenic human anti-acetylcholine receptor (AchR) autoantibody for the induction of experimental autoimmune myasthenia gravis (EAMG) in monkeys. Methods The antigen-binding fragment of anti-AchR antibody (Fab637) isolated from the thymus of myasthenia gravis (MG) was cloned into vector pIgG1 containing immunoglobulin (IgG1) gene to construct a recombinant plasmid that can express intact IgG1 anti-AchR antibody in mammalian cells (IgG1-637) recombinant expression vector (pIgG1-637), the pIgG1-637 transfected CHO-k1 cell line, screened stably expressed IgG1-637 cell strain expanded culture. Purified IgG1-637 was injected into 5 rhesus macaques to determine the pathogenicity of IgG1-637 by detecting monkey muscle contractility and complex muscle movement potential (CMAP) to determine the incidence of monkey EAMG. Results 20μmol / L MSX (L-methionine sulfoxmine) -selected CHO-k1 cells expressed the highest level of antibody IgG1-637 and the expression yield was 17.4μg / ml. Three monkeys (injection doses of 1.7, 1.7 and 5.0 mg / kg, respectively) developed clinical symptoms of muscle weakness within one week after injection of IgG1-637 and showed a decrease in CMAP. Conclusion The complete IgG1-637 constructed by Fab637 is a pathogenic anti-AchR antibody that induces EAMG in monkeys.