Construction of anti-VEGFR-2 IgG1 like human antibody and its expression in CHO-k cells / 药学学报
Acta Pharmaceutica Sinica
;
(12): 1544-1549, 2013.
Artigo
em Chinês
| WPRIM
| ID: wpr-298046
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.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Plasmídeos
/
Proteínas Recombinantes de Fusão
/
Imunoglobulina G
/
Fragmentos Fc das Imunoglobulinas
/
Transfecção
/
Cricetulus
/
Células CHO
/
Receptor 2 de Fatores de Crescimento do Endotélio Vascular
/
Alergia e Imunologia
/
Anticorpos de Cadeia Única
Tipo de estudo:
Estudo prognóstico
Limite:
Animais
Idioma:
Chinês
Revista:
Acta Pharmaceutica Sinica
Ano de publicação:
2013
Tipo de documento:
Artigo
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