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荧光共振能量转移技术阳性参照质粒的构建及应用
Construction of CFP-YFP positive plasmid and its application in fluorescence resonance energy transfer
【摘要】 目的构建青色荧光蛋白(CFP)基因和黄色荧光蛋白(YFP)基因相连接的真核表达载体pYFP-CFP,并检测融合蛋白CFP-YFP的FRET转移效率,为FRET技术提供阳性参照物。方法利用基因重组法,以带有CFP基因序列的质粒(p CFP)为模板,扩增CFP基因并插入黄色荧光蛋白载体(pYFP)的多克隆位点中,构建真核表达质粒pYFP-CFP。经细胞转染后,使其表达CFP和YFP相连的融合蛋白。在激光共聚焦显微镜下采集FRET信号图和FRET转移效率(EFRET)的计算,并比较和分析表达CFP-YFP的阳性细胞与CFP和YFP共表达的阴性细胞。结果真核表达质粒pYFP-CFP构建成功,并在细胞质中表达,这与p CFP和pYFP共转染的细胞中荧光蛋白的分布略有不同,后者在细胞胞质和细胞核中均有表达。统计结果显示表达融合蛋白的阳性组EFRET明显高于共表达的阴性组,差异有统计学意义。结论融合荧光蛋白分子的青色和黄色蛋白之间具有10个氨基酸残基相连,符合FRET现象发生的条件,能够采集到很强的FRET信号,可作为FRET研究的稳定阳性实验参照。
【Abstract】 Objective To construct cyan and yellow fused fluorescence protein( CFP-YFP) and detect its fluorescence resonance energy transfer( FRET) efficiency( EFRET) in cells for providing a positive control of FRET research. Methods Cyan fluorescence protein gene was amplified from p CFP vector by PCR and then inserted into multiple cloning site( MCS) of pYFP vector. The recombinant plasmid pYFP-CFP was transfected into HEK293 cells and expressed CFP-YFP. Using confocal microscopy system,FRET signal and differences of EFRET between the positive control CFP-YFP and the negative control CFP & YFP were detected and analyzed. Results The results of restriction enzyme analysis and nucleotide BLAST identification confirmed that the pYFP-CFP was successfully constructed. The CFP-YFP fused fluorescence protein was expressed in cytoplasm,while the YFP & CFP fluorescence protein was expressed in cytoplasm and nuclear. EFRET was significantly different between positive control and negative control. Conclusion CFP and YFP are linked by 10 amino acid residues,and the strong FRET signal of CFP-YFP can be detected in cells. CFP-YFP is a positive and stable tool for FRET research.
【Key words】 plasmid construction; fluorescence resonance energy transfer; transfer efficiency; CFP-YFP;
- 【文献出处】 山西医科大学学报 ,Journal of Shanxi Medical University , 编辑部邮箱 ,2015年11期
- 【分类号】Q782
- 【被引频次】2
- 【下载频次】221