慢病毒转染荧光蛋白EYFP-H148Q/I152L细胞模型的构建
The cell model establishment through lentivirus transfecting fluorescent protein EYFP-H148Q/I152L

摘要：目的 构建稳定表达EYFP荧光蛋白的HeLa细胞作为阴离子(卤素)通道阻断剂筛选的细胞模型，为高通量筛选阴离子(卤素)通道阻断剂提供条件。方法 通过基因重组技术构建表达YFP突变蛋白(EYFP-H148Q/I152L)和puromycin抗性的慢病毒载体。将慢病毒载体和包装质粒混合物转染包装细胞293T细胞产生慢病毒颗粒，感染HeLa细胞，进一步利用抗性基因对细胞进行puromycin筛选，纯化细胞并扩增至所需细胞量。采用实时定量PCR和Western blot方法检测目的基因EYFP-H148Q/I152L的表达效率。并验证EYFP-HeLa稳转细胞系作为卤族离子通道阻断剂筛选模型的活性。结果 基因测序验证了目的基因成功插入慢病毒载体。RT-PCR和Western blot检测结果显示，目的基因在HeLa细胞中实现了过表达。荧光显微镜下观察到EYFP-HeLa稳转细胞株能够表达特有的EYFP黄色荧光，效率接近100%。碘离子(I-)(低渗)溶液刺激细胞阴离子(卤素)通道的开放，内流的I-能够使黄色荧光淬灭。结论 构建的EYFP-HeLa细胞系能够稳定表达EYFP黄色荧光蛋白，对内流入细胞的I-敏感，可以作为理想的阴离子(卤素)通道阻断剂的筛选模型。
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)