抗CD20嵌合抗体的构建与表达  

Construction and expression of chimeric anti-human CD20 monoclonal antibody

作　　者：王玉刚[1] 黄英[1] 谷欣[1] 于鸣[1] 冯健男[1] 孙瑛勋[1] 黎燕[1] 沈倍奋[1] 

机构地区：[1]军事医学科学院基础医学研究所分子免疫室,北京100850

出　　处：《细胞与分子免疫学杂志》2006年第3期363-367,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(No.30490240)

摘　　要：目的构建抗CD20嵌合抗体的真核表达载体并实现在真核细胞中的表达。方法采用RT-PCR,从分泌鼠源抗人CD20抗体的杂交瘤细胞系1-28中钓取其轻、重链基因,连接至T载体,进行序列测定。还原SDS聚丙烯酰胺凝胶电泳分离抗CD20单克隆抗体(mAb)1-28的轻、重链蛋白,切取轻链及重链条带,质谱测定氨基酸序列,Biolynx和pepeseq软件分析可信度。对比所测DNA序列与蛋白序列,确定所钓取基因的正确性。将mAb1-28轻重链可变区基因,连接至表达载体pCMV-VH和pCMV-VL,构建成嵌合抗体C1-28的轻链真核表达载体C1-28L及重链真核表达载体C1-28H。PCR、酶切及序列测定以确定连入基因的正确性。脂质体介导法将嵌合抗体的轻重链真核表达载体共转染入293T细胞,RT-PCR检测mRNA水平的表达,夹心ELISA法检测蛋白表达量,Westernblot检测目的蛋白的大小。结果钓取到mAb1-28基因。mAb部分蛋白序列测定结果与根据所钓取基因推出的蛋白序列相对应。成功构建了嵌合抗体的真核表达载体,并实现真核表达,表达量可达257mg/L,其相对分子质量(Mr)与人IgG的Mr一致。结论为后续研究嵌合抗CD20抗体对非霍奇金淋巴瘤的治疗提供了一定的依据。AIM： To construct the eukaryotic expression vector of chimeric anti-human CD20 monoclonal antibody （mAb） and realize its expression. METHODS： The light- and heavy-chain genes were amplified from hybridoma cell line 1-28 secreting anti-human CD20 mAb by RT-PCR and were cloned to T vector and sequenced. Proteins of mAb 1-28 were separated by reducing SDS-PAGE. Light- and heavy-chain bands were excised from preparative gel, digested by trypsin, and subjected to peptide mass fingerprinting. Software Biolynx and pepeseq were used to evaluate the score of probability. Correctness of the light- and heavy-chain DNA sequences was verified by their protein sequences. Genes of VH and VL were amplified from T vector and cloned into chimeric antibody expression vector （ pCMV-VH ＆ pCMV-VL）, generating the expression vectors of chimeric anti-human CD20 mAb （C1 -28） including light chain expression vector C1 -28L and heavy chain expression vector C1 -28H. The two plasmids were co-transfected into 293T cells with Lipofectamine^TM 2000. RT-PCR was used to detect the transcription at mRNA level. C1 -28 expression was detected by sandwich ELISA and Western blot methods. RESULTS： mAb 1 - 28＇s genes were successfully cloned and verified by peptide mass fingerprinting. Eukaryotic expression vectors of anti-human CD20 mAb were constructed and expressed in 293T cells with the expression amount reaching 257 mg/L and the molecular weight consistent with that of human IgG. CONCLUSION： These experiments lay solid foundation for further study on the role of chimeric CD20 antibody in the treatment of non-Hodgkin＇s lymphoma.

关 键 词：杂交瘤 质谱分析 嵌合抗体 

分 类 号：R392.11[医药卫生—免疫学]