CaMBP-10单克隆抗体的制备及CaMBP-10在豌豆器官中的表达检测

用电泳纯钙调素结合蛋白BP 10 (CaMBP 10 )免疫小鼠 ,制备单克隆抗体 (McAb) .用MEP(mercapto ethtyl pyridine)HyperCel疏水层析柱从细胞培养上清中纯化并获得单克隆抗体 ,同时测定了抗体 抗原反应的基本特性 .此单克隆抗体具有较高纯度、特异性和亲和力 .亲和常数 (Kaff)为1 2 6× 10 9(mol L) -1,此抗体和CaM在空间上以相同或相近的位点与CaMBP 10相结合 .以胶体金标记的抗体为探针 ,研究CaMBP 10在豌豆幼叶、成熟叶、茎尖、茎、根等不同器官的分布特征 ,并与胶体金标记CaM的结合情况相对照 .结果显示 ,CaMBP 10在植物中的分布特点与文献报道的CaM的分布特点相一致 ,提示CaMBP 10可能是在蛋白水平上对CaM进行区域化和可用性调节

Development of the Monoclonal Antibody Against CaMBP-10 and the Detection on CaMBP-10 Expression in Different Pea Organs

The monoclonal antibody against calmodulin binding protein BP 10 (CaMBP 10)was developed with purified CaMBP 10. The antibody was purified with MEP(mercapto ethtyl pyridine)HyperCel column from cell culture supernatants and characterized. The results indicated that the antibody showed high purity, specificity and affinity. Affinity constants to coated antigen was 1 26×10 9(mol/L) -1 , and it binds CaMBP 10 at the same or close site with calmodulin. Using this antibody, the distributional feature of CaMBP 10 in young leaf, leaf, stem apex, stem and root of pea was studied , and compared with the result of gold labeled calmodulin. It was showed that CaMBP 10 had the same distributional feature as that of calmodulin, which indicated that probably CaMBP 10 regulated calmodulin on compartmentalization and availability at the protein level.