定点突变去除抗乙酰胆碱受体抗体的补体结合功能_临床医学论文.docVIP

定点突变去除抗乙酰胆碱受体抗体的补体结合功能_临床医学论文 定点突变去除抗乙酰胆碱受体抗体的补体结合功能_临床医学论文 【摘要】 ［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. 【关键词】 受体，胆碱能；重症肌无力；点突变；补体结合；抗体 ABSTRACT: 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. METHODS A pathogenic anti AChR antibody IgG 637, previously made in our laboratory, was mutated in the key site of complement C 1 q binding K 322 A by site directed mutagenesis technology. The genes of mutant IgG 637/K 322 A were then transformed into E coli XL 1 Blue for cloning, sequencing and furthermore transinfected into mammalian cell line CHO k 1 for expression. The complement C 1 q-binding activity of expressed products IgG 637/K 322 A was determined in enzyme linked immunosorbent assay (ELISA) by using pig anti human C 1 q monoclonal antibody. RESULTS The mutant IgG 637/K 322 A was not able to bind complement C 1 q in ELISA, whereas, the control antibody IgG 637 could bind C 1 q 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 IgG 637 by site directed mutagenesis. Key words:receptor, cholinergic;myasthenia gravis;point mutation;complement-binding;antibodies 重症肌无力是由抗乙酰胆碱受体抗体介导的自身免疫病，而致病性抗乙酰胆碱受体抗体多为IgG类抗体，它的2个抗原结合片段（Fab）与神经肌肉接头处突触后膜上相邻的2个乙酰胆碱受体结合，可加快乙酰胆碱受体的内化作用（即抗原调变）；也可通过其可结晶片段（Fc）激活补体的C 1 q成分，经过级联反应形成C 5-C 9膜攻击复合体，导致乙酰胆碱受体被破坏，出现肌肉收缩无力等症状［1］.由致病性抗乙酰胆碱受体抗体制备的单链抗体因具有单价结合性，不能引起抗原调变，又因缺乏Fc段而不能激活补体，但仍能与乙酰胆碱受体结合，从而封闭致病性抗体与乙酰胆碱结合，对乙酰胆碱受体具有保护作用，可用于重症肌无力的治疗［2］.但单链抗体分子质量小，稳定性差，在应用前需进行基因工程加工处理，以延长在血中的半寿期.随着分子生物学技术的飞速发展和抗体工程