西安交通大学学报（医学版）2016，Vol.37Issue(4)：478-483,6.DOI:10.7652/jdyxb201604003
慢病毒转染荧光蛋白 EYFP-H148Q/I152 L细胞模型的构建
The cell model establishment through lentivirus transfecting fluorescent protein EYFP-H148Q/I152L
摘要
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) .关键词
阴离子/卤素/荧光蛋白/阴离子通道阻断剂/筛选模型/基因重组Key words
anion/halide ion/fluorescence protein/anion channel blocker/screening model/gene recombination分类
医药卫生引用本文复制引用
肖远,许荣,李秀敏,刘艳,黎星,霍聪,王晓明..慢病毒转染荧光蛋白 EYFP-H148Q/I152 L细胞模型的构建[J].西安交通大学学报（医学版）,2016,37(4):478-483,6.基金项目
军队保健计划课题（No ．13BJZ34）及卫生部行业基金（No ．201302008）资助Supported by the Health Care Plan of the Army （No ．13BJZ34） and the Industry Fund of Ministry of Health （）
