title: [Construction of anti-VEGFR-2 IgG1 like human antibody and its expression in CHO-k cells].
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.
authors.affiliation: State Key Laboratory of Natural Medicines, College of Life Science and Technology, China Pharmaceutical University Nanjing 210009, China.
authors.forename: Zhi-Ke
authors.initials: ZK
authors.lastname: Li
authors.affiliation: State Key Laboratory of Natural Medicines, College of Life Science and Technology, China Pharmaceutical University Nanjing 210009, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: He
authors.affiliation: State Key Laboratory of Natural Medicines, College of Life Science and Technology, China Pharmaceutical University Nanjing 210009, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural Medicines, College of Life Science and Technology, China Pharmaceutical University Nanjing 210009, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Natural Medicines, College of Life Science and Technology, China Pharmaceutical University Nanjing 210009, China.
authors.forename: Wan-Lu
authors.initials: WL
authors.lastname: Cao
authors.affiliation: State Key Laboratory of Natural Medicines, College of Life Science and Technology, China Pharmaceutical University Nanjing 210009, China.
authors.forename: Ze-Gen
authors.initials: ZG
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Natural Medicines, College of Life Science and Technology, China Pharmaceutical University Nanjing 210009, China.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000032:analysis*; D016466:CHO Cells; D003412:Cricetulus; D005822:Genetic Vectors; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D057127:Single-Chain Antibodies / Q000235:genetics; D014162:Transfection; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000276:immunology*
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D040301:Vascular Endothelial Growth Factor Receptor-2