一种新型β_1肾上腺素受体单克隆抗体的制备及其临床应用

A new monoclonal antibody against beta1-adrenergic receptor and its clinical application

目的:采用杂交瘤细胞融合技术构建特异性针对β_1肾上腺素受体(β_1-AR)细胞外第二环的单克隆抗体(β_1-AAmAb)。方法:设计合成两条肽段,偶联后作为免疫原混合免疫小鼠,挑选免疫反应强的小鼠进行细胞融合。筛选杂交瘤细胞、扩大培养,并于接种小鼠,14d后收集腹水,用Protein G对腹水进行纯化以获得单克隆抗体。结果:ELISA结果显示,杂交瘤细胞上清液中β_1-AA的滴度明显高于对照组(2.9896±0.1873)vs.(0.0635±0.0386),差异有统计学意义(P<0.001)。Western bolt分析发现,β_1-AAmAb与商业化抗β_1-AR多克隆抗体阳性对照具有相同分子量的条带。激光共聚焦结果表明,β_1-AAmAb可与天然存在的β_1-AR相结合。Annexin V/PI流式分析发现,β_1-AAmAb可以诱导心肌细胞发生早期凋亡。乳鼠心肌细胞跳动频率实验证实,β_1-AAmAb与心力衰竭患者血清中的β_1-AA均可增加乳鼠心肌细胞跳动频率。结论:本研究成功构建了与心力衰竭患者血清中存在的β_1-AA具有相同生物学效应的单克隆抗体,使用该单克隆抗体进行后续的研究可以使我们对β_1-AA的致病机制有更好的认识。

Objective： To construct the monoclonal antibody against the extracellular second loop of β_1-adrenergic receptor（ β_1-AAmAb）,using hybridoma fusion technique. Methods： Blood samples were collected from 18 heart failure patients as well as 10 healthy subjects,and β_1-AA was detected using ELISA. Two peptides were coupled to keyhole limpet hemocyanin（ KLH）. Mice were immunized with 2 fusion peptides.Splenocytes of immunized mice were fused with myeloma cell line. Positive hybridoma clones were cultivated.Two mice were vaccinated with hybridoma cells secreting β_1-AAmAb. After 14 days,ascitic fluid from immunized mice was collected. Ig Gs were purified via protein G antibody affinity chromatography. Results： Compared to the control,β_1-AA titer was markedly increased in sera of heart failure patients（ 0. 643 ± 0. 086） vs.（ 0. 155 ± 0. 084,P〈0. 001） and the hybridoma supernatant[（ 2. 9896 ± 0. 1873） vs.（ 0. 0635 ± 0. 0386,P〈0. 01） respectively. Western blot analysis showed β_1-AAmAb had the same band as commercial anti-β_1-AR polyclonal antibody. Moreover,β_1-AAmAb bonded with native β_1-ARs expressed on cardiomyocytes,and increased cardiomyocyte beat frequency. Annexin V and PI assay demonstrated that β_1-AAmAb induced cardiomyocytes apoptosis. Conclusion： In this study,a monoclonal antibody exerting the same biological effects asβ_1-AAs circulating in patients with heart failure was constructed successfully. Themethod ology provides a reliable strategy for exploring the pathogenesis of β_1-AA.