重症肌无力治疗性单价IgG抗体基因的构建  

Construction of a monovalent IgG antibody gene for therapy of myasthenia gravis

作　　者：李钟燮[1] 孟繁平[1] 孙昌元[1] 李芳芳[1] 金权鑫[1] 李红花[1] 李英信[1] M.Stassen M.de Baets 

机构地区：[1]延边大学基础医学院免疫学与病原生物学教研部,延吉133000 [2]Department of Neurology,Faculty of Medicine,University of Maastricht

出　　处：《中国免疫学杂志》2009年第11期1003-1005,共3页Chinese Journal of Immunology

基　　金：国家自然科学基金资助项目(No.30760234;30860260)

摘　　要：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。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/ CDR3LXPLKP were then transformed respectively into E. coli XL1-Blue for cloning, sequencing and furthermore transinfected into mammalian cell line CHO-kl 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/CDR3△PLKP 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 IgC.637/K322A/CDR3LXPLKP was not able to hind the specific antigen human AChR in RIA. The correct mutations of knob and hole were observed by sequencing. Conclusion： The genes of monovalent IgG antiAChR antibody without complement-binding activity are successfully developed from a pathogenic anti-AChR antibody IGG637.

关 键 词：重症肌无力 单价抗体 定点突变 免疫球蛋白G 

分 类 号：R392.11[医药卫生—免疫学]