大容量人天然抗体库的构建、鉴定及初步应用  

Construction, Characterization and Preliminary Application of a Large Naive Human Antibody Library

作　　者：唐晓明[1] 王清明[1] 杨俊涛[1] 陈吉中[1] 范国才[1] 汪思应[2] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850 [2]安徽医科大学,合肥230032

出　　处：《中国生物工程杂志》2005年第10期17-24,共8页China Biotechnology

摘　　要：从未经主动免疫的健康志愿者的外周血淋巴细胞中提取总RNA,用RT-PCR扩增人抗体重链(VH)和轻链(VL)可变区基因,得到了6种VH家族基因,11种VL家族基因,这些抗体基因家族覆盖了人抗体基因多样性的95%以上。采用改进的SOEPCR法将VH基因和VL基因连接成人单链抗体(scFv)基因,并克隆到噬菌粒载体pCANTAB5E中,将连接产物电转化大肠杆菌TG1,经辅助噬菌体M13KO7超感染,构建了库容为5.58×109的噬菌体单链抗体库。采用BstNI酶切法证明,构建的噬菌体单链抗体库具有良好的多样性。以TNF-α为靶,从该抗体库中筛选到了抗TNF-α抗体,这说明该抗体库可用于人源抗体的筛选。Total RNA was extracted from the peripheral blood lymphocytes of the non-immunized healthy donors ,and used to amplified human V genes by RT-PCR. Six VH germline genes and eleven VL germline genes were successfully amplified. They cover more than 95% of the human antibody used. Genes encoding single-chain Fv fragments were asembled by improved SOE PCR,and cloned into the phagemids （pCANTABSE）. The recombinant phagemids were transformed into the competent E. coli TG1 cells. A large human antibody library containing 5.58 × 10^9 clones was created after rescuing the recombinant phagemids from the transformed E. coli TG1 cells. This library exhibited high diversity as judged by the BstN I restriction pattern. The scFv antibodies against TNF-α were successfully selected from the library by panning. This meant that the library would be useful for generating human antibodies by-passing immunization.

关 键 词：容量 人天然抗体库 构建方法 主动免疫 外周血 淋巴细胞 噬菌体 抗体药物 生物技术 

分 类 号：TQ464[化学工程—制药化工]