Expression and purification of CDNF and preparation of its polyclonal antibodies
10.3969/j.issn.1000-484X.2015.09.016
- VernacularTitle:多巴胺能神经营养因子真核表达、纯化及多克隆抗体制备
- Author:
Lizheng WANG
;
Zixuan WANG
;
Rui ZHU
;
Zhentian LIU
;
Bin YU
;
Xianghui YU
;
Xinghong ZHAO
- Publication Type:Journal Article
- Keywords:
CDNF;
Polyclonal antibody;
Ni-NTA immunoaffinity chromatography;
Parkinson′s disease
- From:
Chinese Journal of Immunology
2015;(9):1221-1224
- CountryChina
- Language:Chinese
-
Abstract:
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.
