利用SPR检测26肽与β_1-肾上腺素受体自身抗体的相互作用

Detection of the interaction between the peptide and β_1-adrenergic receptor autoantibodies using surface plasmon resonance

目的探讨根据β_1肾上腺素受体细胞外第二环(β_1-adrenergic receptor,β_1-AR-EC_Ⅱ)的氨基酸序列合成的26肽与β_1-肾上腺素受体β_1-AR-EC_Ⅱ的单克隆抗体(β_1-adrenergic receptor autoantibodies,β_1-AA)的相互作用。方法根据人β_1-肾上腺素受体细胞外第二环(β_1-AR-EC_Ⅱ)的氨基酸序列合成26肽;采用杂交瘤细胞融合的方法获得针对β_1-AR-EC_Ⅱ的单克隆抗体(β_1-AA);利用表面等离子共振(surface plasmon resonance,SPR)检测该合成的26肽与β_1-AA的亲和力;利用乳鼠心肌细胞跳动频率实验验证该合成26肽对β_1-AA的中和作用。结果提取的β_1-AA可以使乳鼠心肌细胞跳动频率增加(P<0.05),提示该β_1-AA具有生物学活性;合成26肽与β_1-AA结合的亲和力(K_D)为4.44μmol/L,属于中等强度结合;较β_1-AA组相比,26肽处理后可明显降低心肌细胞跳动频率(P<0.05)。结论该合成26肽与β_1-肾上腺素受体自身抗体之间存在相互作用,并且可以拮抗β_1-AA引起的乳鼠心肌细胞跳动频率的增加。

Objective To investigate the interaction between the peptide which mimic the structure of the second extracellular loop of the β1-adrenergic receptor（ β1-AR-ECⅡ） and β1-adrenergic receptor autoantibodies（ β1-AA）. Methods Peptide was synthesized according to the amino acid sequence of human β1-AR-ECⅡ; a hybridoma fusion method was used to obtain anti-β1-AR-ECⅡmonoclonal antibody; the interaction between the peptide and β1-AA was detected by surface plasmon resonance（ SPR）; and the beating frequency experiment of neonatal rat cardiomyocytes was explored to test the neutralization of the peptide to β1-AA. Results β1-AA enhanced the beating frequency of neonatal rat cardiomyocytes（ P〈0. 05）,indicating that the β1-AA we obtained has biological activity; SPR results showed that there was a moderate binding affinity（ KD） of 4. 44 μmol/L between the synthetic peptide and β1-AA. Conclusion There was interaction between the synthesized peptide and β1-adrenergic receptor autoantibodies.