小鼠源性ABIN1蛋白的抗体制备及功能验证  

Preparation of polyclonal antibodies against mABIN1 protein and its biological activity identification

作　　者：赵钧如[1] 李琳琳[1] 周培岚[1] 宫泽辉[1] 

机构地区：[1]军事医学科学院毒物药物研究所,北京100850

出　　处：《军事医学》2013年第8期582-586,共5页Military Medical Sciences

基　　金：北京市自然科学基金资助项目(7092078);国家自然科学青年基金资助项目(30901799)

摘　　要：目的制备小鼠源性A20结合核因子κB活化抑制蛋白1(A20 binding inhibitor of NF-κB activation 1,mABIN1)的多克隆抗体并进行功能验证,利用该抗血清对大鼠脑区ABIN1的分布进行验证。方法利用mABIN1片段与钥孔血蓝蛋白(keyhole limpet hemocyanin,KLH)耦联得到的肽段为免疫原,免疫新西兰大耳白兔3次,共35 d,获得抗血清,分别用ELISA、蛋白免疫(Western)印迹和免疫组织荧光化学法对抗体的特异性进行验证;使用上述验证所得的特异性抗血清,通过Western印迹检测ABIN1在大鼠各脑区的分布;对所得到的特异性抗血清进行纯化。Western印迹证明,纯化后的抗体杂带较纯化前明显降低,但效价也有所下降。结果与结论成功制备了兔抗小鼠ABIN1抗血清并对抗血清进行纯化,经该抗血清首次验证ABIN1在大鼠嗅球、皮质、海马、纹状体、伏隔核、丘脑、下丘脑、小脑、脑干及脊髓等中枢神经系统不同部位均有广泛分布,为在动物体内进行ABIN1生物学功能研究提供了有利工具。Objective To establish polyclonal antibodies of mouse A20 binding inhibitor of NF-κB activation 1（mABIN1） and identify its biological activity and the distribution of ABIN1 in the rat brain.Methods Three peptides of mABIN1 coupled with keyhole limpet hemocyanin（KLH） were immunized to New Zealand rabbits about 35 days for three times.The specificity of antiserum against mABIN1 was characterized by ELISA,Western blotting and immunofluorescence histochemistry.The distribution of ABIN1 in the rat brain was studied by the polyclonal antiserum of mABIN1 using Western blotting.Western blotting showed that there was a lower hetero-band in the brain tissues of the polyclonal antibody purified from antiserum than in the antiserum of mABIN1.Results and Conclusion The successful preparation of polyclonal antibody of mABIN1 will provide efficient tools in the studies of ABIN1 in animal experiments.For the first time,ABIN1 is proved to be distributed in the central nervous system including the olfactory bulb,prefrontal cortex,hippocampus,striatum,nucleus accumbens,thalamus,subthalamus,cerebellum,brainstem and spinal cord in rats by Western blotting.

关 键 词：小鼠源性ABIN1 钥孔血蓝蛋白 耦联肽 抗体 纯化 

分 类 号：R392.11[医药卫生—免疫学]