Construction of cell model targeted on the damage by α-synuclein
10.3969/j.issn.1001-1978.2015.04.029
- VernacularTitle:建立以α-突触核蛋白损伤为靶点的细胞筛选模型
- Author:
Wenfen YAN
;
Yang HENG
;
Qianhang SHAO
;
Naihong CHEN
;
Yuhe YUAN
- Publication Type:Journal Article
- Keywords:
α-synuclein;
toxic damage;
Parkinson’s Disease;
screen model;
protein expression;
drug target
- From:
Chinese Pharmacological Bulletin
2015;(4):586-590
- CountryChina
- Language:Chinese
-
Abstract:
Aim To construct the cell model targeted on the damage by α-synuclein for screening anti-Parkinson’s Disease (PD)compounds.Methods The cDNA fragment of α-synucle-in gene was obtained by PCR methods and inserted into the re-combinant prokaryotic plasmid by molecular cloning technique. The recombinant plasmid was transformed into Escherichia coli, and subsequently induced to express α-synuclein protein.The recombinant α-synuclein was purified and identified by affinity chromatography,immunoblotting and mass spectrometry.The cells damage by α-synuclein was evaluated through cell viability measured by 3-(4,5-dimethyl-2-thiazolyl )-2,5-diphenyl-2-H-tetrazolium bromide.Results The obtained cDNA fragment ofα-synuclein in accordance with its theoretic molecular weight was cloned into pET30a plasmid and verified by sequencing.The re-combinant plasmid was transformed into bacteria E.Coli.BL21 (DE3)and induced to express α-synuclein by isopropyl β-D-1 -thiogalactopyranoside (IPTG).The expression condition was op-timized according to the culture temperature,the concentration of IPTG and the proliferation state of bacteria.The purified α-synu-clein was proved to be a 1 5.3 ku molecule weight protein,and could be immunoblotted with anti-α-synuclein antibody.The pu-rified α-synuclein could decrease the viability of PC1 2 cells and primary neurons significantly,and its effect was in a concentra-tion-dependent manner.Conclusion We have succeeded in constructing the cell model targeted on the damage by α-synucle-in.
