多巴胺能神经营养因子真核表达、纯化及多克隆抗体制备  

Expression and purification of CDNF and preparation of its polyclonal antibodies

作　　者：王立正 王子璇[1] 朱瑞[1] 刘镇天 于彬[1] 于湘晖[1] 赵兴红[1] 

机构地区：[1]吉林大学生命科学学院艾滋病疫苗国家工程实验室,长春130012

出　　处：《中国免疫学杂志》2015年第9期1221-1224,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金项目(31300756;81472816);吉林省科技发展计划青年科研基金项目(20140520007JH)资助

摘　　要：目的:获得具有活性的脑多巴胺能神经营养因子(CDNF)蛋白并制备CDNF多克隆抗体。方法:选用VR1012作为真核表达载体,在HEK293T细胞中表达带有his标签的CDNF蛋白,镍柱亲和层析进行纯化,SDS-PAGE检测纯化情况,Western blot鉴定蛋白的正确性,MTT检测CDNF蛋白对PC12细胞的保护活性。免疫新西兰大白兔,制备多克隆抗体。结果:构建的p VR1012-CDNF-his在HEK 293T细胞中高效分泌表达,镍柱亲和层析获得纯度90%以上的CDNF蛋白,MTT结果显示纯化获得的蛋白具有对PC12细胞的保护作用。免疫动物后,成功制备出CDNF多克隆抗体。结论:通过HEK293T细胞中表达-镍柱亲和层析获得具有纯度较高且具有生物活性的CDNF蛋白,并成功制备多克隆抗体以方便后续对于CDNF应用及相关机制的研究。Objective： To obtain purified and functional CDNF-his recombinant protein and prepare its polyclonal antibodies.Methods：Preparation of recombinant CDNF-his was carried out in HEK 293 T cells with pVR1012-CDNF-his successfully constructed transfected into them.Then,the recombinant protein was purified by Ni-NTA immunoaffinity chromatography.The purity was analyzed by SDS-PAGE and the protein′s identity was tested by Western blot.MTT was used to verify the biological function of the protein purified.New Zealand white rabbits were immunized with purified CDNF-his protein for preparation of polyclonal antibodies.Results：pVR1012-CDNF-his expressed successfully in HEK 293 T cells.The purity of protein was up to more than 90%after purification.MTT showed that CDNF-his was able to protect PC 12 cells from damage by 6-OHDA.The polyclonal antibody was detected at the end of animal immunizing process.Conclusion： A method to express and purify protein using HEK 293T cell and following Ni-NTA immunoaffinity chromatography has been built.CDNF-his with biological activity is obtained based that.Finally, polyclonal antibodies of CDNF were generated successfully.

关 键 词：CDNF 多克隆抗体 镍柱亲和层析 帕金森病 

分 类 号：R392.33[医药卫生—免疫学]