CAJ | 학술논문

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TR 1-404抗体蛋白功能特性.方法构建pcDNA3.4载体抗体质粒,转染Expi293 FTM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TR1-404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Expi293 FTM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.
목적 건립고효표체전인원항인종류배사인자상관유도조망배체수체(TRAIL)-R1단극륭항체(TR1-404)단백체계,병평개TR 1-404항체단백공능특성.방법 구건pcDNA3.4재체항체질립,전염Expi293 FTM세포,표체항체단백.수집세포배양상청,사용ProteinA/G순화항체단백.이용쌍항체협심화간접매련면역흡부시험(ELISA)검측항체단백농도급특이성.류식세포분석검측TR1-404여Hela세포화HCT116세포표면TRAIL-R1적결합능력.MTT법검측재불동시간점TR1-404、TRAIL급이자연합작용대Hela세포화HCT116세포생존화증식적영향.Annexin V/PI쌍염색검측Hela세포화HCT116세포조망.결과 성공구건pcDNA3.4재체항체질립화건립Expi293 FTM세포단백고효표체계통.정제적TR1-404항체기여중조TRAIL-R1항원특이성결합,야능여Hela화HCT116세포표면적TRAIL-R1결합.발현TR1-404연합TRAIL작용우Hela세포,종류세포조망효과현저제고.이재HCT116세포중,TRAIL여TR1-404적연합작용미견현저제고.결론 성공건립항체단백고효표체계통.TR1-404항체특이성여TRAIL-R1항원결합.TR1-404항체제고TRAIL유도적Hela세포조망작용.본연구위TRAIL-R1파향적항종류치료연구제공신방법.
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 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.