抗肝癌细胞噬菌体单链抗体库的构建及筛选  

Construction and Screening of Anti-Hepatoma Cells Phage Display scFv Library

作　　者：粘红[1] 安利国[2] 赵岩[3] 陈吉中[1] 范国才[1] 王清明[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850 [2]山东师范大学生命科学院,济南250014 [3]吉林大学生命科学学院,长春130023

出　　处：《现代免疫学》2004年第5期357-361,共5页Current Immunology

摘　　要：构建抗人肝癌细胞单链抗体库 ,从中筛选与肝癌细胞特异结合的高亲和力单链抗体。从HepG2细胞免疫的BALB/c小鼠脾脏提取总RNA ,RT PCR扩增小鼠抗体重、轻链可变区基因 ,用 (Gly4Ser) 3 连接肽基因 ,经重叠延伸反应 ,在体外将VH 和VL 连接成单链抗体 (scFv)基因 ,并克隆入噬菌粒载体pCANTAB5E中 ,构建噬菌体单链抗体库。以HepG2细胞为抗原对抗体库进行淘选 ,ELISA法鉴定各单克隆与肝癌细胞的结合活性 ,并对阳性克隆进行表达。成功构建了库容为 1 1× 10 6抗肝癌细胞的噬菌体单链抗体库 ,经筛选得到了与HepG2细胞具有较强结合能力的单链抗体 ,实现了scFv在大肠杆菌中的可溶性表达。序列测定结果表明 ,VH 和VL 基因符合小鼠抗体可变区特征 。To obtain the hepatoma cell-specific antibody, a phage display antibody library was constructed. The total RNA was isolated from spleens of BALB/c mice immunized with Hep G2 cells, and was used to amplify V H and V L genes by RT-PCR. V H and V L genes were joined with a DNA linker encoding peptide(Gly 4Ser) 3 as a sin gle chain variable fragments(scFv). Then, scFv fragments were cloned into phagemids(pCANTAB5E), and the recombinant phagemids were used to transform E.coli TG1 to construct phage antibody libr ary. The library was screened with Hep G2 cells, and the positive recombinant phages identified by ELISA were used to infect E.coli TOP10 for the production of soluble scFv antibodies. The results showed that a phage display antibody library containing 1.1×106 different clones was obtained. The scFvs with good affinity to hepatoma cells were screened. Western blot showed that scFvs were successfu lly expressed in E.coli TOP10. Sequencing indicated that V H and V L genes were homologous with the variable region of mouse antibody.

关 键 词：肝癌细胞 单链抗体 SCFV 噬菌体抗体库 筛选 

分 类 号：Q78[生物学—分子生物学]