重症肌无力治疗性单价IgG抗体基因的构建

摘要

Objective:To construct a monovalent IgG antibody gene for specific immunotherapy of myasthenia gravis.Methods:A pathogenic anti-human acetylcholine receptor (AChR) antibody IgG637,previously developed in our laboratory,was mutated in the site of K322A to remove the complement binding activity,and 4 amino acids (PLKP) were deleted in complemetary determining region 3 (CDR3) to remove the specific antigen binding activity by site-directed mutagenesis technology.The genes of mutant IgG637/K322A and IgG637/K322A/CDR3ΔPLKP were then transformed respectively into E.coli XL1-Blue for cloning,sequencing and furthermore transinfected into mammalian cell line CHO-k1 for expression.The complement C3-binding activity of expressed product IgG637/K322A was determined in ELISA,and the human AChR-binding activity of IgG637/K322A/CDR3ΔPLKP in RIA.The mutant antibody genes were further undergone mutation of knob (T366Y) and hole (Y407T) to favor the heterodimerization of H chain in making monovalent IgG antibody.Results:The mutant IgG637/K322A was not able to bind complement C3 in ELISA,and the mutant IgG637/K322A/CDR3ΔPLKP was not able to bind the specific antigen human AChR in RIA.The correct mutations of knob and hole were observed by sequencing.Conclusion:The genes of monovalent IgG anti-AChR antibody without complement-binding activity are successfully developed from a pathogenic anti-AChR antibody IgG637.%目的:构建一株对重症肌无力(MG)具有特异性免疫治疗作用的单价IgG类抗体基因.方法:应用定点突变技术,将致病性抗乙酰胆碱受体(AChR)抗体IgG637的重(H)链第322位氨基酸进行K322A突变,获得的突变型抗体IgG637/K322A基因再进行H链互补决定区3(CDR3)缺失突变,获得突变型抗体IgG637/K322A/CDR3ΔPLKP基因.经转化大肠杆菌XL1-Blue进行增殖后,转染哺乳类细胞CHO-k1进行表达,表达产物经ELIAS检测与补体C3的结合活性,经RIA检测与特异性抗原人AChR的结合活性.突变抗体H链再经杵(T366Y)臼(Y407T)突变,以利于异源H链的配对.结果:突变型抗体IgG637/K322A丧失了与补体C3结合的能力,突变型抗体IgG637/K322A/CDR3ΔPLKP丧失了与人AChR结合的能力.测序证实已经获得了预想的杵臼突变序列.结论:已经成功的制备了无补体激活能力的单价IgG类抗AChR抗体基因.