中文摘要：

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 （1 μg·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。

英文摘要：

Anti-angiogenesis mechanism plays a vital role in tumor targeting immunotherapy. Based on the amino acid sequence of an anti-VEGFR-2 scFv-Fc fusion antibody （AK404R-Fc）, this article is aimed to generate an anti-VEGFR-2 human IgG1-like full length antibody （Mab-04）. Firstly, the light chain （L-chain） and heavy chain （H-chain） were obtained by overlap PCR and then linked to eukaryotic expression vector pcDNA3.1, separately. The recombinant plasmids （pcDNA3.1-L-chain and pcDNA3.1-H-chain） were then co-transfected into CHO-k cells using liposome transient transfection. Subsequently, Mab-04 antibody was expressed and purified by Protein A affinity chromatography. Western blotting was applied to identify the expression of Mab-04 and its affinity was detected by ELISA assay. DNA sequencing revealed the successful construction of recombinant plasmids and Western blotting assay proved the successful expression of full-length antibody （1 μg·mL-1）. Finally, ELISA assay illustrated that the binding of the antibody to its antigen was in a concentration-dependent manner （IC50： 50 nmol·L-1）. These outcomes above indicated that Mab-04 was successfully expressed and assembled, which laid the foundation for further preparation and antineoplastic activity study.