基因工程乳腺癌特异性人单链抗体的表达与特性  

Expression and characterization of single chain antibody derived from a breast cancer specific human monoclonal antibody hybridoma CM-1 cell line

作　　者：张云[1] 王树蕙[1] 郭彩云[1] 李文辉[1] 扬帆[1] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所微生物及病原学室,北京100005

出　　处：《基础医学与临床》2002年第4期324-327,329,共5页Basic and Clinical Medicine

基　　金：卫生部基金 (96 10 6 7)

摘　　要：研究解决分泌人单抗的杂交瘤细胞系难于稳定持久分泌抗体的难题 ,制备单链抗体 ,使单抗分子小型化 ,为进一步研究其在肿瘤诊断和治疗中的应用作准备。从分泌抗乳腺癌人单抗的杂交瘤细胞CM 1总RNA中 ,利用RT PCR技术分别扩增出人单抗重链可变区VH 基因和轻链可变区VL 基因 ,将扩增产物纯化后克隆于pGEM T载体中 ,进行DNA测序和序列比较分析后 ,将两者共克隆于表达载体中诱导表达 ,利用斑点免疫印迹及竞争抑制法检测表达产物的抗原性。所克隆的CM 1人单抗重链可变区和轻链可变区基因片段 ,分别属于人免疫球蛋白IgMⅢ亚群 ,和鼠κ轻链V亚群 ,ⅩⅦ家族 ,用斑点免疫印迹法检测可见表达产物能与人乳腺癌细胞特异结合 ;人CM 1单克隆抗体对此单链抗体与人乳腺癌细胞的结合有竞争性抑制作用 ,抑制率为 75 7%。结论成功地制备了可特异结合乳腺癌细胞的CMHuman monoclonal antibody hybridoma derived single chain antibodies(scFvs) hold great potential in the cancer immune imaging diagnosis and target therapy. CM 1 is a breast cancer specific human monoclonal antibody hybridoma cell line. To obtain CM 1 scFv for diagnosis and treatment of breast cancer the total RNA of CM 1 was isolated ,the variable fragment regions of heavy strain and the light strain were amplified by RT PCR and then inserted into pGEM T vector. DNA sequencing showed that V H fragment originated from human subtype and the V L fragment from mouse subtype. The coding sequence was then subcloned into expression vector pWAI180. The recombinant plasmid was transformed into E.coli Gm2163, scFv expressed after induction with IPTG. CM 1 scFv bound specifically to breast cancer CAM cell line as demonstrated by Immune dot blot assay. Competitive ELISA showed that 75.0% binding activity of CM 1 ascite was inhibited by the CM 1 scFv.

关 键 词：基因工程 乳腺癌 人单克隆抗体CM-1 单链抗体 斑点免疫印迹 竞争抑制法 

分 类 号：R737.9[医药卫生—肿瘤] Q789[医药卫生—临床医学]