Construction of anti-VEGFR-2 IgG1 like human antibody and its expression in CHO-k cells.
- Author:
Zhike LI
;
Yuan HE
;
Juan ZHANG
;
Wei XIE
;
Wanlu CAO
;
Zegen WANG
;
Min WANG
- Publication Type:Journal Article
- From:
Acta Pharmaceutica Sinica
2013;48(10):1544-9
- CountryChina
- Language:Chinese
-
Abstract:
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 microg x mL(-1)). Finally, ELISA assay illustrated that the binding of the antibody to its antigen was in a concentration-dependent manner (IC50: 50 nmol x 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.
