人源性天然抗体库的构建及淀粉样蛋白Ab1-42寡聚体单链抗体的筛选和鉴定

Isolation and identification of a human single chain Fv antibody against amyloid-beta 1-42 soluble oligomers from a human phage display library

摘要:

本研究旨在利用噬菌体展示技术构建人源性天然抗体库, 以可溶性Ab1-42寡聚体对抗体库进行筛选获得针对低分子量Ab1-42寡聚体的特异性单链抗体。利用RT-PCR法从10个健康人外周血淋巴细胞中得到全套人抗体VH和VL基因, 经过重叠延伸PCR将VH和VL连接得到scFv片段, 将scFv片段酶切后克隆至pCANTAB5E噬菌体载体, 电转化TG1感受态菌, 获得库容为2.5×109单链抗体库。经辅助噬菌体M13K07拯救, 以可溶性Ab1-42寡聚体为抗原, 对抗体库进行4轮筛选, ELISA法筛选特异性识别Ab1-42寡聚体的阳性克隆, 将筛选到的阳性克隆B19转化至E. coli HB2151菌, 诱导表达可溶性scFv抗体。SDS-PAGE及Western blotting分析结果显示可溶性scFv抗体获得了正确表达, 且能够与Ab1-42三聚体及纤维特异性结合, 亲和力(Kd)为9×10−6 mol/L。Ab1-42寡聚体特异性单链抗体的获得为老年性痴呆症(AD)的治疗研究奠定了基础。

Abstract:

To get specific scFv (Single-chain fragment variable) antibody against soluble Ab1-42(Amyloid-beta) oligomers, we constructed a human single-chain Fv (scFv) antibody library by phage display technology. Using RT-PCR, we amplified the variable heavy (VH) and variable light (VL) genes from peripheral blood lymphocytes (PBL). Then we obtained the scFv fragments through SOE-PCR, and the scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent Escherichia coli TG1 cells. Consequently, a scFv phage display library containing 2.5×109 clones was constructed. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for Ab1-42 oligomers were enriched after four rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. Positive clone B19 was used to infect E. coli HB2151 to express soluble scFv antibody. SDS-PAGE and Western blotting analysis showed that the soluble scFv B19 antibody was expressed successfully and could bind specifically to Ab1-42 trimer and protofiber. The specific scFv against Ab1-42 oligomers can be used in the therapeutic research on Alzheimer’s disease.