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目的构建抗CD40嵌合抗体的真核表达载体,实现在真核细胞中的高效表达。方法采用RT-PCR法,从分泌鼠源抗人CD40抗体的杂交瘤细胞系5C11中钓取其轻、重链基因,进行序列测定。将5C11轻重链可变区基因分别克隆入表达载体pCMV-VH和pCMV-VL,构建成嵌合抗体(c5C11)的轻链真核表达载体5C11L-pCMV及重链真核表达载体5C11H-pCMV。将嵌合抗体的轻重链真核表达载体共转染入293T细胞,利用夹心ELISA法测定上清中c5C11的浓度,通过流式细胞术检测c5C11与膜抗原的特异性结合。通过生物信息学相关方法对c5C11的氨基酸序列进行合理改造,尝试在保持生物学功能的基础上提高c5C11的表达量。结果成功钓取鼠源单抗5C11的轻重链可变区基因并构建了真核表达载体,实现了c5C11的分泌表达;嵌合抗体较好地保留了鼠源母本抗体的抗原识别能力。利用计算机辅助设计,一定程度上提高了c5C11的表达量。结论为后续研究嵌合抗CD40抗体对B淋巴瘤等肿瘤的治疗提供了一定的依据。
Objective To construct the eukaryotic expression vector of anti-CD40 chimeric antibody to achieve high expression in eukaryotic cells. Methods The light and heavy chain genes of the hybridoma cell line 5C11 secreting murine anti-human CD40 antibody were screened by RT-PCR and sequenced. The 5C11 light and heavy chain variable region genes were cloned into the expression vectors pCMV-VH and pCMV-VL respectively to construct the light chain eukaryotic expression vector 5C11L-pCMV of chimeric antibody (c5C11) and the heavy chain eukaryotic expression vector 5C11H-pCMV. The light and heavy chain eukaryotic expression vectors of chimeric antibody were co-transfected into 293T cells. The concentration of c5C11 in the supernatant was determined by sandwich ELISA. The specific binding of c5C11 to membrane antigen was detected by flow cytometry. Through the bioinformatics-related methods, the amino acid sequence of c5C11 was modified reasonably to try to improve the expression of c5C11 on the basis of maintaining the biological function. Results The light and heavy chain variable region genes of murine monoclonal antibody 5C11 were successfully obtained and eukaryotic expression vector was constructed to achieve the secretory expression of c5C11. The chimeric antibody retained the antigen recognition ability of murine maternal antibodies. The use of computer-aided design, to a certain extent, increased the expression of c5C11. Conclusion This study provides some evidences for further research on the treatment of tumors such as B lymphoma with chimeric anti-CD40 antibody.