抗VEGFR-2全人源IgG1抗体的构建及其在CHO-k细胞中的表达  

Construction of anti-VEGFR-2 IgG1 like human antibody and its expression in CHO-k cells

作　　者：李致科[1] 何远[1] 张娟[1] 解伟[1] 曹婉璐[1] 王泽根[1] 王旻[1] 

机构地区：[1]中国药科大学天然药物活性组分与药效国家重点实验室,生命科学与技术学院,江苏南京210009

出　　处：《药学学报》2013年第10期1544-1549,共6页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81072561;81102364);天然药物活性组分与药效国家重点实验室资助项目(JKGP201101);江苏省青蓝工程项目(2010)

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(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。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.

关 键 词：VEGFR-2 全长抗体 抗肿瘤血管生成 

分 类 号：R963[医药卫生—微生物与生化药学]