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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 prepare and characterize a mouse polyclonal antibody against A20 binding inhibitor of NF-κB activation 1 (mABIN1). The distribution of ABIN1 in rat brain regions verification. Methods Immunized New Zealand white rabbits with the peptide of mABIN1 and keyhole limpet hemocyanin (KLH) three times for 35 days to obtain antiserum. ELISA and protein immunization Western blot and immunofluorescence were used to verify the specificity of the antibody. Using the specific antiserum verified above, the distribution of ABIN1 in various brain regions of rats was detected by Western blotting. The obtained specific antiserum Purification. Western blot showed that the purified antibody bands were significantly lower than before purification, but the titer also decreased. RESULTS AND CONCLUSION: Rabbit anti-mouse ABIN1 antiserum was successfully prepared and purified. The antiserum was used for the first time to verify the effect of ABIN1 in olfactory bulb, cortex, hippocampus, striatum, nucleus accumbens, thalamus, hypothalamus, cerebellum, Brainstem and spinal cord and other central nervous system are widely distributed in different parts of the body for the study of ABIN1 biological function provides a useful tool.