定点突变去除抗乙酰胆碱受体抗体的补体结合功能

Removal of complement binding activity of an anti-acetylcholine receptor antibody by site directed mutagenesis

[目的]制备无补体结合功能的免疫球蛋白样抗乙酰胆碱受体抗体,用于重症肌无力的特异性免疫治疗.[方法]应用定点突变技术,将致病性抗乙酰胆碱受体抗体IgG 637的重链CH 2区的第322位氨基酸进行K 322 A突变,获得的突变型抗体IgG 637/K 322 A基因经转化大肠杆菌XL 1-Blue进行增殖,测定序列证实为突变序列,转染哺乳类细胞CHO-k 1进行表达,其产物经酶联免疫吸附试验检测与补体C 1 q的结合活性.[结果]突变型抗乙酰胆碱受体抗体IgG 637/K 322 A不能与补体C 1 q结合,而对照抗体IgG 637可与补体结合.[结论]经定点突变,成功地获得了失去补体结合功能的抗乙酰胆碱受体抗体.

OBJECTIVE To develop an immunoglobulin-like anti acetylcholine receptor（AChR） antibody without complement binding activity,which can be used in the specific immunotherapy for myasthenia gravis.METHODSA pathogenic anti AChR antibody IgG637,previously made in our laboratory,was mutated in the key site of complement C1q binding K322A by site directed mutagenesis technology.The genes of mutant IgG637/K322A were then transformed into E coli XL1 Blue for cloning,sequencing and furthermore transinfected into mammalian cell line CHO k1 for expression.The complement C1q-binding activity of expressed products IgG637/K322A was determined in enzyme linked immunosorbent assay（ELISA） by using pig anti human C1q monoclonal antibody.RESULTSThe mutant IgG637/K322A was not able to bind complement C1q in ELISA,whereas,the control antibody IgG637 could bind C1q in a concentration dependent manner.CONCLUSION The immunoglobulin like anti AChR antibody without complement binding activity is successfully made from a pathogenic anti AChR antibody IgG637 by site directed mutagenesis.