抗CD20嵌合抗体的表达与活性检测  

Construction, Expression and Characterization of Anti-CD20 Chimeric Monoclonal Antibody

作　　者：师明磊[1] 胡显文[1] 陈惠鹏[1] 高丽华[1] 李世崇[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《中国生物工程杂志》2005年第7期34-39,共6页China Biotechnology

摘　　要：表达了基因重组抗CD20嵌合抗体并对其生物学活性进行了初步鉴定。设计合成轻、重链可变区序列;提取血液RNA,通过RT-PCR得到人κ、IgG1的轻、重链恒定区序列。运用重叠延伸PCR,连接可变区与恒定区,将轻、重链基因连接至pIRES双表达载体。将质粒以阳离子脂质体转染CHO细胞,ELISA挑选阳性克隆,共获得7株表达较高的克隆,表达量约为2mg/L。扩大培养阳性克隆anti-CD20-1B3,收获上清,以蛋白A进行亲和层析纯化表达蛋白。SDS-PAGE检测表明纯化纯度达到95%,蛋白相对分子量与理论值吻合。以CD20+细胞Raji、Daudi、Ramous检测,表明该抗体能与CD20抗原特异性结合,体外杀伤试验说明抗体能够杀伤CD20+淋巴瘤细胞。A chimeric antibody against CD20 on B cell was constructed and expressed in CHO cell and its characters was studied. VH and VL genes were designed according to reported sequence, they were synthesised as oligonucleotide and then connected by overlap extension PCR; CH(IgG1)and CL(κ) genes were amplified from normal B cell RNA by RT-PCR. They were connected by overlap extension PCR and then cloned into pIRES bicistronic expression vector. The vector was transfected into CHO cell for expression. High expression level clones were selected by ELISA. 7 clones were found to secrete antibody productively. The expression product of anti-CD20-1B3 was purified by proteinA affinity chromatography which specifically bind to Fc fragment of antibody. Protein concentration was measured by ultraviolet spectrometer. The expression concentration of 1B3 was about 2mg/L. Reduced SDS-PAGE analysis showed that the relative molecular weight of chimeric antibody was coincidence with expectation, its purity was about 95 % . ELISA assay revealed that the chimeric antibody being capable of binding specifically to human lymphoma cell lines(Raji, Ramous, Daudi) expressing CD20 antigen. Cytotoxicity experiment revealed that the chimeric antibody alone induced direct cytotoxicity in CD20-expressing human lymphoma cell line.

关 键 词：嵌合抗体 活性检测 SDS-PAGE检测 RT-PCR 阳性克隆 生物学活性 重链可变区 双表达载体 CHO细胞 脂质体转染 ELISA 相对分子量 特异性结合 初步鉴定 基因重组 设计合成 IGG1 重链基因 扩大培养 层析纯化 RNA 阳离子 表达量 

分 类 号：Q78[生物学—分子生物学] Q786