The cell model establishment through lentivirus transfecting fluorescent protein EYFP-H148Q/I152L
10.7652/jdyxb201604003
- VernacularTitle:慢病毒转染荧光蛋白 EYFP-H148Q/I152 L细胞模型的构建
- Author:
Yuan XIAO
;
Rong XU
;
Xiumin LI
;
Yan LIU
;
Xing LI
;
Cong HUO
;
Xiaoming WANG
- Publication Type:Journal Article
- Keywords:
anion;
halide ion;
fluorescence protein;
anion channel blocker;
screening model;
gene recombination
- From:
Journal of Xi'an Jiaotong University(Medical Sciences)
2016;37(4):478-483
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish the HeLa cell line that can stably express EYFP fluorescent protein as the model for anion channel blocker (halide ion) screening ,which lays the foundation for high throughput screening of anion channel blocker (halide ion) .Methods Through gene recombination technology ,a new lentivirus vector which can express mutant protein YFP (EYFP‐H148Q/I152L) and puromycin resistance ,was built .The mixture of lentivirus vector and packaging plasmid was transfected into 293T cells to produce lentivirus particles . After infection of HeLa cells by the lentivirus particles ,puromycin was used to screen the cells as YFP‐positive HeLa cell line .Then cell amplification was carried out after purification and efficiency of EYFP‐H148Q/I152L was further detected by Real‐time quantitative PCR (RT‐PCR) and Western blot .We then verified the activity of EYFP‐HeLa transfected cell line as a screening model of anion channel blocker .Results Gene sequencing verified that EYFP‐H148Q/I152L was successfully inserted into lentivirus vectors .RT‐PCR and Western blot results showed that the target gene was overexpressed in HeLa cells . The specific yellow fluorescence of EYFP of HeLa cells could be observed under fluorescence microscope with the efficiency of nearly 100% . I- (low permeability ) solution stimulated the opening of anion (halogen) channels ,and the yellow fluorescence was quenched by I - flow into cells . Conclusion The EYFP‐HeLa cell line can stably express EYFP yellow fluorescent protein and is sensitive to the internal flow of I - .Therefore ,it can be used as an ideal screening model of anion channel blocker (halide ion) .
