Construction and expression of anti-tumor necrosis factor related apoptosis-inducing ligand receptor death receptor 5 chimeric antibody in eukaryotic cells.
- Author:
Feng CHEN
1
;
Ya-Bin GUO
;
Shi-Lian LIU
;
De-Xian ZHENG
;
Yan-Xin LIU
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Antibodies; genetics; immunology; pharmacology; Antineoplastic Agents; immunology; pharmacology; CHO Cells; Cell Survival; drug effects; Cricetinae; Cricetulus; Gene Expression; Humans; Mice; Protein Engineering; Receptors, TNF-Related Apoptosis-Inducing Ligand; genetics; immunology; Recombinant Fusion Proteins; genetics; immunology; pharmacology
- From: Acta Academiae Medicinae Sinicae 2008;30(6):690-695
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct the human/mouse chimeric antibody of a functional anti-death receptor 5 (DR5) antibody. Methods The viable region of light chain (VL) and viable region of heavy chain (VH) genes of anti-DR5 antibody were amplified and cloned into the light- and heavy-chain expression vectors respectively, then the recombinant plasmids were co-transfected into dihydrofolate reductase(-) Chinese hamster ovary cell (CHO-dhfr(-)) for expression. The positive clone was screened by the two selective genes (neo and dhfr). The humanization and specificity of chimeric antibody was identified by ELISA and Western blotting, and the tumoricidal activity of the expressed chimeric antibody was detected by tetrazolium salt phenazine methosulfate assay.
RESULTSThe expression vectors stably expressed chimeric antibody in CHO-dhfr(-). In the cell supernatant of the F4' clone, the human IgG heavy constant region and light constant region were identified. Moreover, the secreted chimeric antibody retained the binding capacity to the antigen (DR5) and decreased the cell viability of Jurkat and HCT116 cells to 73.15% and 77.30% in vitro respectively.
CONCLUSIONThe human/mouse anti-DR5 chimeric antibody has been successfully expressed in eukaryotic cells and shows tumoricidal activity, which establishes a foundation for the future research of humanized antibody medicine.
