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Insight Ⅱ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-EGFP/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 primarily identify the antigenic epitopes of agonist type anti-CD40 monoclonal antibody, 5C11, which was constructed in our previous research, by means of computer modeling and site-directed mutation experiments. Methods:The structures of antigen and antibody were modeled by Insight Ⅱ software and the immune complex was constructed. The antigenic epitope of 5C11 antibody was calculated and speculated. The full length of wide type human CD40 (wtCD40) gene and two site-directed mutant CD40 gene (70muCD40 and 114muCD40) were amplified by RT-PCR; pIRES2-EGFP/wtCD40, pIRES2-EGFP/70muCD40 and pIRES2-EGFP/114muCD40 recombinant vectors were constructed. These vectors were transfected into HEK293 cells by Lipofect method, and HEK293 cells stably transfected with pIRES2-EGFP/wtCD40, pIRES2-EGFP/70muCD40 and pIRES2-EGFP/114muCD40 vectors (named HEK293/wtCD40, HEK293/70muCD40 and HEK293/114muCD40 cells, respectively) were screened. The binding abilities of HEK293/wtCD40, HEK293/70muCD40 and HEK293/114muCD40 cells with 5C11 were examined by flow cytometry and Western blotting analysis. Results: The recombinant eukaryotic expression vectors pIRES2-EGFP/wtCD40, pIRES2-EGFP/70muCD40 and pIRES2-EGFP/114muCD40 were successfully constructed and the corresponding stably transfected HEK293 cells were obtained. The binding ability between 5C11 antibody and HEK293/70muCD40 and HEK293/114muCD40 cells were lower than that with HEK293/wtCD40 cells. Western blotting results showed that 5C11 antibody only recognized HEK293/wtCD40 cells but not HEK293/70muCD40 and HEK293/114muCD40 cells. Conclusion: The 70th threonine and 114th glutamic acids in human CD40 amino acid sequence are the antigenic epitopes of 5C11 monoclonal antibody, which has potential clinical significance for humanized CD40 antibody research.

重大新药创制科技重大专项资助项目（No. 2009zx09103），教育部创新团队资助项目（No. IRT0849），国防预研资助项目（No. B3820110002）