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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 class antibody gene with specific immunotherapy for myasthenia gravis (MG). Methods: Mutation of K322A at amino acid 322 of heavy chain (H) of pathogenic AChR antibody IgG637 by site-directed mutagenesis. The mutant IgG637 / K322A mutant was further subjected to H chain complementarity determining region 3 (CDR3) deletion mutation to obtain the mutant antibody IgG637 / K322A / CDR3ΔPLKP gene. The recombinant plasmid was transformed into E.coli XL1-Blue and then transfected into mammalian cells CHO-k1 for expression. The expressed product was tested for its binding activity to complement C3 by ELIAS, and the binding activity to specific antigen human AChR was detected by RIA. The mutant antibody H chain was further mutated through a pestle (T366Y) cell (Y407T) to facilitate pairing of heterologous H chains. Results: Mutant antibody IgG637 / K322A lost its ability to bind to complement C3, and mutant antibody IgG637 / K322A / CDR3 ΔPLKP lost its ability to bind human AChR. Sequencing confirmed that the expected pestlar mutation sequence was obtained. CONCLUSION: Monovalent IgG anti-AChR antibody genes without complement activation have been successfully prepared.