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.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2005年第10期17-24,共8页
China Biotechnology
