特异性哇巴因结合肽的筛选及其结合活性的测定

Selection of specific ouabain conjugated peptides and identification of its binding activity

目的 本研究的目的在于寻找特异性哇巴因结合肽(Ouabainconjugatedpeptide,OCP) ,降低哇巴因水平。 方法 利用噬菌体随机肽库表面呈现技术筛选出OCP ,通过测序 ,分析 ,获得氨基酸序列 ,合成筛选出的 12肽 ,采用放射性配基受体结合法 (radioligandbindingassay ,RRA)检测哇巴因结合肽与哇巴因的结合能力。结果 从噬菌体 12肽库中筛选出 3种多肽 ,其中肽A(12肽 )的筛选一致率达到 0 6 7(8/12 )。RRA实验结果显示 ,合成的肽与3 H ouabain具有一定的结合能力 ,平衡解离常数为 1 0 87nmol·L-1,受体密度为12 0pmol·g-1Pro ,IC50 =1 4 6× 10 -4mol·L-1,而且两者之间的结合无相互协同作用。结论 特异性OCP的获得 ,不仅获得了与哇巴因特异结合的蛋白质序列 。

AIM To select specific ouabain conjugated peptides (OCP) to decline ouabain level. METHODS Biopanning phage displayed 12 peptide library for OCP. The sequences of each selected peptide was determined and analyzed. The binding activity of synthetical OCP was identified by radioligand binding assay (RRA). RESULTS Three kinds of peptides were found out. Peptide A (12 peptide) was occupied in 0 67(8/12). The analysis of protein showed that there was no homogeneous protein in Genebank. The results of RRA showed that there were binding activity between synthetical OCP and 3H ouabain. Dissociation constant(K d) was 1 087 nmol·L -1 . Receptor density was 120 pmol·g -1 protein. IC 50 =1 46×10 -4 mol·L -1 . CONCLUSION It is important to obtain distinctive OCP, by which not only the protein sequence was analyzed, but also a valuable experimental data in detection of ouabain and therapy in hypertension also was offered.