抗人CD40功能性嵌合抗体的制备及其高效表达  

Preparation and enhanced eukaryotic expression of anti-human CD40 antibodies

作　　者：化晓莉[1,2] 乔春霞[2] 黎燕[2] 张学光[1] 

机构地区：[1]苏州大学医学生物技术研究所,江苏苏州215007 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《军事医学科学院院刊》2010年第3期225-229,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：国家高技术研究发展计划(863计划)(2006AA02A254);国家自然科学基金青年科学基金(30901381)

摘　　要：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 expression vector of chimeric anti-human CD40 monoclonal antibodies（mAb）and obtain functional chimeric antibodies in the eukaryotic system.Methods The light-chain（VL）and heavy-chain（VH）genes were obtained from hybridoma cell line 5C11 secreting anti-human CD40 mAb by RT-PCR and subcloned to T vector for sequencing.Genes of VH and VL were then cloned to vectors pCMV-VH and pCMV-VL,respectively,to construct the expression vector pair of chimeric 5C11（c5C11）,including light chain expression vector 5C11L-pCMV and heavy chain expression vector 5C11H-pCMV.The two plasmids were co-transfected into 293T cells,and the omcentration of c5C11 in supernatant was detected by sandwich ELISA.Membrane antigen binding specificity was analyzed by flow cytometry.Then the amino acid sequence of 5C11 was analyzed and optimized in order to enhance the expression level of soluble c5C11.Results The genes of 5C11＇s light and heavy chain variable domain were cloned successfully.Eukaryotic expression vectors were constructed and humanized antibody c5C11 was harvested in the supernatant of transfected 293T cells,which maintained a satisfactory antigen binding ability.By computer-aided design,high expression level of modified antibody could be detected.Conclusion This study offers useful information for further a high studyies on the role of chimeric anti-CD40 antibody in the treatment of malignancy such as B lymphoma.

关 键 词：CD40 抗体 单克隆 杂交瘤 嵌合抗体 真核表达 

分 类 号：R392.11[医药卫生—免疫学]