Wilson蛋白N端多克隆抗体的制备和纯化

Preparation and Purification of Polyclonal Antibody Against N-Terminal of ATP7B

【目的】制备并纯化Wilson蛋白(ATP7B)的N端多克隆抗体,为进一步研究其基因表达蛋白的结构和功能打下基础。【方法】RT-PCR扩增目的基因,GST基因融合载体表达融合蛋白,亲和层析进行蛋白纯化,Thrombin酶切并收集目的蛋白,免疫家兔,ELISA检测抗体滴度,离子交换层析纯化抗体血清,Westernblot检测抗体特异性。【结果】ELISA方法检测抗体滴度达到了1∶2500,Westernblot表明该抗体有很好的特异性,非变性SDS-PAGE显示纯化后的抗体IgG纯度很高。【结论】应用该方法成功制备了Wilson病蛋白质量的多克隆抗体。

To prepare and purify specific antibody against N Terminal of ATP7B(Wilson Protein),and to make a good foundation for study of the structure and function of ATP7B. The target gene was amplified by RT PCR. Fusion protein was expressed with GST gene fusion system. Affinity chromatography was used for protein purification. Thrombin was used to cleave fusion protein with on column cleavage method. The aimed protein was gathered as antigen. Rabbits were immunized subcutaneously. The titre of the antibody was detected by ELISA assay. The antiserum was purified with DEAE Sephadex A 25 columns. Western blot was applied to confirm the specificity of the antibody.ELISA assay revealed that the titer of the prepared antiserum against fusion protein was as high as 1∶2 500. Western blot showed that the antibody was able to react with the proteins. Non denatured SDS PAGE confirmed high purity of IgG. [Conclusion]Polyclonal antibody against ATP7B,is successfully prepared.