Establishment of a high efficient expression system of a novol human monoclonal antibody against tumour necrosis factor-related apoptosis-inducing ligand receptor and its functional characteristics

Objective To establishe a high efficient expression system and to generate mAb to tumour necrosis factor (TNF)-related apoptosis-inducing ligand receptor-1(TRAIL)-R1, and to evaluate its functional characteristics . Methods We constructed a pcDNA3.4 vector containing anti-TRAIL-R1 Ab gene, and established a high efficient method expressing Ab protein using Expi293F™ cell expression system.We then purified TR1-404 Ab using Protein A/G.We detected the production of TR1-404 and its specific binding activity by the double antibody sandwich enzyme-linked immunosorbent assay(ELISA) and indirect ELISA .TRAIL-R1 expression on Hela cells and HCT116 cells was determined by flow cytometric assay.The survival and proliferation of Hela cells and HCT116 cells were analyzed after treatment with TR1-404 alone or TRAIL alone or combination of TR1-404 with TRAIL using MTT assay.We measured TR1-404 or TRAIL-induced tumor cell apoptosis using Annexin Ⅴ/PI double staining. Results We successfully constructed plasmid expressing mAb to TRAIL-R1 and established a high efficient protein expression system.TR1-404 can bind to not only recombinant human TRAIL-R1 specifically but also TRAIL-R1 on surface of Hela and HCT116 cells.We found that TR1-404 enhanced TRAIL-induced cell apoptosis in Hela cells but not in HCT116. Conclusions In the study, we successfully establish a high protein expression system.TR1-404 can bind to TRAIL-R1 specifically.TR1-404 Ab increased TRAIL-mediated apoptosis in Hela tumor cells. Key words: Tumour necrosis factor related apoptosis-inducing ligand; Tumour necrosis factor related apoptosis-inducing ligand receptor-1; Monoclonal antibody; Apoptosis