目的:通过计算机模拟与点突变实验初步探讨本课题组前期研制的抗人CD40激发型单克隆抗体5C11识别的抗原表位。方法:利用InsightⅡ软件分别模拟抗原、抗体结构,构建抗原抗体复合物模型,通过计算推测5C11单抗所识别的抗原表位。构建人野生型CD40(wtCD40)及其第70位苏氨酸突变型(70muCD40)和第114位谷氨酸突变型(114muCD40)的重组真核表达载体pIRES2-EGFP/wtCD40、pIRES2-EGFP/70muCD40和pIRES2-EGFP/114muCD40,脂质体转染法将重组载体导入HEK293细胞,筛选稳定转染细胞株(即HEK293/wtCD40、HEK293/70muCD40和HEK293/114muCD40细胞)。流式细胞术和Western blotting检测5C11单抗与HEK293/wtCD40、HEK293/70muCD40和HEK293/114muCD40细胞的结合能力。结果:成功构建pIRES2-EG-FP/wtCD40、pIRES2-EGFP/70muCD40和pIRES2-EGFP/114muCD40真核表达载体和相应稳定转染细胞株。5C11单抗与HEK293/70muCD40和HEK293/114muCD40细胞结合能力较HEK293/wtCD40细胞明显减弱;Western blotting检测结果表明,5C11单抗仅识别HEK293/wtCD40细胞,不识别HEK293/70muCD40和HEK293/114muCD40细胞。结论:人CD40氨基酸序列的第70位苏氨酸和第114位谷氨酸是其单抗5C11识别的抗原表位,对构建人源化CD40抗体具有潜在的临床意义。 OBJECTIVE: To preliminary explore the antigenic epitopes recognized by anti-human CD40-stimulated monoclonal antibody 5C11 developed by our research group through computer simulation and point mutation experiments. Methods: Using Insight Ⅱ software to simulate the antigen and antibody structure, respectively, to construct the antigen-antibody complex model, and to deduce the epitope recognized by 5C11 monoclonal antibody. The recombinant eukaryotic expression vectors pIRES2-EGFP / wtCD40, pIRES2-EGFP / 70muCD40 of human wild type CD40 (wtCD40) and its 70th threonine mutation (70muCD40) and 114th glutamate mutant (114muCD40) And pIRES2-EGFP / 114muCD40. Lipofectamine was used to transfect the recombinant vector into HEK293 cells. The stably transfected cell lines (HEK293 / wtCD40, HEK293 / 70muCD40 and HEK293 / 114muCD40 cells) were screened. The binding ability of 5C11 McAb to HEK293 / wtCD40, HEK293 / 70muCD40 and HEK293 / 114muCD40 cells was detected by flow cytometry and Western blotting. Results: The eukaryotic expression vector pIRES2-EG-FP / wtCD40, pIRES2-EGFP / 70muCD40 and pIRES2-EGFP / 114muCD40 were successfully constructed and the corresponding stable transfected cell lines were successfully constructed. The binding ability of 5C11 McAb to HEK293 / 70muCD40 and HEK293 / 114muCD40 cells was significantly weaker than that of HEK293 / wtCD40 cells. The results of Western blotting showed that 5C11 mAb only identified HEK293 / wtCD40 cells but not HEK293 / 70muCD40 and HEK293 / 114muCD40 cells. CONCLUSION: The 70 th amino acid and the 114 th glutamic acid of human CD40 amino acid sequence are epitopes recognized by the monoclonal antibody 5C11, which has potential clinical significance for the construction of humanized anti-CD40 antibody.