全人源抗人肿瘤坏死因子相关诱导凋亡配体受体单克隆抗体蛋白高效表达体系的建立及其功能性分析

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

目的建立高效表达全人源抗人肿瘤坏死因子相关诱导凋亡配体受体（TRAIL）-R1单克隆抗体（TR1-404）蛋白体系，并评价TR1-404抗体蛋白功能特性。方法构建pcDNA3．4载体抗体质粒，转染Expi293F^TM细胞，表达抗体蛋白。收集细胞培养上清，使用ProteinA／G纯化抗体蛋白。利用双抗体夹心和间接酶联免疫吸附试验（ELISA）检测抗体蛋白浓度及特异性。流式细胞分析检测TRl_404与Hela细胞和HCT116细胞表面TRAIL-R1的结合能力。MTT法检测在不同时间点TR1-404、TRAIL及二者联合作用对Hela细胞和HCT116细胞生存和增殖的影响。Annexin V／PI双染色检测Hela细胞和HCT116细胞凋亡。结果成功构建pcDNA3．4载体抗体质粒和建立Expi293F^TM细胞蛋白高效表达系统。精制的TR1-404抗体既与重组TRAIL-R1抗原特异性结合，也能与Hela和HCT116细胞表面的TRAIL-R1结合。发现TR1-404联合TRAIL作用于Hela细胞，肿瘤细胞凋亡效果显著提高。而在HCT116细胞中，TRAIL与TR1-404的联合作用未见显著提高。结论成功建立抗体蛋白高效表达系统。TR1-404抗体特异性与TRAIL-R1抗原结合。TR1-404抗体提高TRAIL诱导的Hela细胞凋亡作用。本研究为TRAIL—R1靶向的抗肿瘤治疗研究提供新方法。

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 Expi293FTM 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 ex- pression on Hela cells and HCT116 cells was determined by flow cytometric assay. The survival and proliferation of Hela cells and HCT116 ceils were analyzed after treatment with TR1-404 alone or TRAIL alone or combina- tion of TR1-404 with TRAIL using MTF 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 HCTll6 cells. We found that TR1-404 en- hanced TRAIL-induced cell apoptosis in Hela cells but not in HCTll6. 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.