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.
Article
en Zh
| WPRIM
| ID: wpr-298046
Biblioteca responsable:
WPRO
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:
1
Índice:
WPRIM
Asunto principal:
Plásmidos
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Proteínas Recombinantes de Fusión
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Inmunoglobulina G
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Fragmentos Fc de Inmunoglobulinas
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Transfección
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Cricetulus
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Células CHO
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Receptor 2 de Factores de Crecimiento Endotelial Vascular
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Alergia e Inmunología
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Anticuerpos de Cadena Única
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
Zh
Revista:
Acta Pharmaceutica Sinica
Año:
2013
Tipo del documento:
Article