抗VEGFR-2 scFv-Fc融合抗体的构建及其在CHO细胞中的稳定表达

Construction of Anti-VEGFR-2 scFv-Fc Fusion Antibody and Stable Expression in CHO-k Cells

目前报道的靶向VEGF/VEGFR基因工程抗体多为不含Fc片段的Fabs、cFv等小分子抗体,该文旨在CHO-k细胞中表达抗VEGFR-2 scFv-Fc融合抗体。将抗VEGFR-2 scFv(AK404)基因与含有人IgG1恒定区的真核表达载体pcDNA3.1-Fc连接,重组质粒测序后经脂质体介导转染CHO-k细胞,G418加压筛选获取稳定表达细胞株。RT-PCR、western blot检测目的基因的转录、表达。测序结果表明重组质粒构建成功,RT-PCR及western blot显示目的基因成功整合到宿主基因组并表达。最终获得可稳定表达抗VEGFR-2 scFv-Fc融合抗体的重组CHO-k细胞株,为融合抗体的大量制备和活性研究打下基础。

Most of the reported engineering antibodies that target VEGF/VEGFR signaling pathway are truncated ones,such as Fab and scFv,which lack Fc fragment.The purpose of this article was to produce anti-VEGFR-2 scFv-Fc fusion antibody in CHO-k cells.Firstly,anti-VEGFR-2 scFv（AK404） gene was ligated with eukaryotic vector pcDNA3.1-Fc which yielded pcDNA3.1-scFv-Fc.After DNA sequencing,CHO-k cells were transfected with the recombinant plasmid by lipofectin according to the manufacturer＇s protocol.Then the stable expression cells were obtained by stressing selection with G418,and transcription and expression were examined by RT-PCR and Western blot respectively.DNA sequencing confirmed that the recombinant plasmid was successfully constructed.RT-PCR and Western blot showed that the target gene was integrated to the genome of the host cells and expressed accordingly.Finally,a recombinant CHO cell line that can express anti-VEGFR-2 scFv-Fc fusion antibody was obtained and propagated stably to generation 25.This study gives solid evidences to following researchers in pilot studies.