【摘要】 目的用人抗乙酰胆碱受体(AchR)自身抗体的抗原结合片段(Fab)构建完整的抗人AchR自身抗体,为重症肌无力(MG)的实验研究提供人源性抗体材料。方法应用PCR技术从分离自MG患者胸腺的Fab637扩增重链Fd片段基因和轻链基因,并克隆至含有免疫球蛋白(IgG1)基因的载体pIgG1上,构建人源性抗AchR自身抗体哺乳类细胞表达的重组载体pIgG1-637。将pIgG1-637转染CHO-k1细胞,细胞培养上清液经斑点杂交试验检测抗体的表达,以放射免疫测定法测定表达的抗体与特异性人AchR结合的活性。结果细胞培养24h后能检测到抗体的分泌,表达的抗体能特异性地与抗原结合,且结合活性很高。结论已成功地构建了具有与抗原特异性结合的人源性IgG1类抗人AchR自身抗体IgG1-637。更多还原

【Abstract】 Objective To construct an intact human anti-acetylcholine receptor (AchR) autoantibody from a human antigen binding fragment (Fab) of anti-AchR antibody for the experimental research of myasthenia gravis (MG). Methods The genes of Fd fragment and light chain of Fab637 from anti-AchR antibody derived from the thymus of patients with MG were amplified by PCR, and then cloned into mammalian expression vector pIgG1 containing IgG1 gene for constructing a recombinant vector pIgG1-637. Furthermore, the pIgG1-637 was transfected into CHO-k1 cell line, and the supernatant of cell culture was checked for the expression of antibodies by dot blot assay. The specific binding activity of the expressed antibodies to human AchR was determined by radioimmunoassay. Results The secretion of the antibodies could be found at 24 h after the transfection. The expressed antibodies were able to bind to human AchR with high specificity and binding activity. Conclusion An intact IgG1 human anti-AchR autoantibody (IgG1-637) with high binding activity is successfully constructed.更多还原