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本文在实验室构建的单链抗体-Fc融合抗体[scFv(AK404R)-Fc]的基础上构建抗VEGFR-2全人源IgG1样全长抗体(Mab-04)。利用重叠PCR,获得Mab-04的轻链和重链的核酸序列后分别克隆到真核表达载体pcDNA3.1,获得重组质粒。脂质体法将重组质粒转染至CHO-k细胞,经Protein A柱纯化细胞培养上清液获得目的蛋白,利用Western blotting检测目的蛋白,ELISA检测Mab-04与抗原亲和力。测序表明重组质粒构建成功,Western blotting检测显示目的蛋白成功表达(1μg·mL-1),ELISA检测阐明该抗体能与抗原结合并呈浓度依赖性(IC50为50 nmol·L-1),表明Mab-04成功表达并正确装配,为进一步大量制备该抗体及其活性研究打下基础。
In this study, a full-length anti-VEGFR-2 IgG1-like antibody (Mab-04) was constructed based on the single-chain antibody-Fc fusion antibody [scFv (AK404R) -Fc] constructed in laboratory. The nucleic acid sequences of the light chain and the heavy chain of Mab-04 were obtained by overlapping PCR and cloned into the eukaryotic expression vector pcDNA3.1 respectively to obtain the recombinant plasmid. The recombinant plasmid was transfected into CHO-k cells by lipofectamine. The recombinant protein was purified by protein A column, and the target protein was obtained by Western blotting. The affinity of Mab-04 to antigen was detected by ELISA. Sequencing showed that the recombinant plasmid was successfully constructed, and the target protein was successfully expressed (1μg · mL-1) by Western blotting. ELISA showed that the antibody could bind antigen in a concentration-dependent manner (IC50: 50 nmol·L-1) -04 was successfully expressed and correctly assembled, which laid the foundation for further mass production of the antibody and its activity.