利用拆分绿色荧光蛋白检测端粒酶TERT亚基与端粒末端蛋白TPP1的相互作用

Using Split Green Fluorescent Protein to Detect the Interaction Between Telomerase TERT Subunit and Telomere Terminal Protein TPP1

目的:构建pCDH-Flag-NGFP-TERT、pCDH-Myc-TPP1-CGFP基因的真核表达载体,验证TPP1-CGFP蛋白能募集到端粒区,并通过GFP蛋白自发重建检测TERT与TPP1的相互作用。方法:以相应质粒为模板,采用聚合酶链反应(polymerase chain reaction,PCR)技术扩增出NGFP、CGFP、TPP1的CDS区编码序列。通过酶切、重组将NGFP插入到pCDH-Flag-TERT载体上,将TPP1-CGFP插入到pCDH-Myc-POT1-v5tag载体上。经菌液PCR、载体酶切及测序验证后转染到293T细胞中,采用蛋白质印迹法检测其表达,免疫荧光和端粒Fish验证TPP1-CGFP能募集到端粒区,将TPP1-CGFP与NGFP-TERT共转重新组装成GFP蛋白验证其相互作用。结果:双酶切结果显示载体构建成功;Western blot结果显示蛋白质成功表达;免疫荧光及端粒Fish显示TPP1-CGFP能募集到端粒区,质粒共转能重新组装成GFP蛋白说明两者有相互作用。结论:真核表达载体构建成功,并证明TPP1-CGFP能募集到端粒区,且拆分后的GFP蛋白能由连接蛋白的相互作用而重新组装并发绿色荧光,为研究端粒酶与TPP1蛋白及端粒的相互作用提供了可视化的细胞模型。

Objective:To construct the eukaryotic expression vector of NGFP-TERT and TPP1-CGFP genes,then verify whether TPP1-CGFP protein can be recruited to the telomere region,and observe the interaction between TERT and TPP1 through the spontaneous reconstruction of GFP protein.Methods:Using the corresponding plasmid as a template,the coding sequences of CDS regions of NGFP,CGFP and TPP1 were amplified by polymerase chain reaction(PCR)technology.NGFP was inserted into the pCDH-Flag-TERT vector by restriction digestion and recombination,and TPP1-CGFP was inserted into the pCDH-Myc-POT1-v5 tag vector.After bacterial liquid PCR,vector enzyme digestion and sequencing verification,it was transfected into 293 T cells,and its expression was detected by Western blot.Immunofluorescence and telomere Fish were used to verify whether TPP1-CGFP can be recruited to the telomere region.Plasmid co-transformation verified that it can be reassembled into GFP protein.Results:The results of double enzyme digestion showed that the NGFP-TERT and TPP1-CGFP vectors were successfully constructed;the plasmid was extracted and transfected into 293 T cells,and Western blot showed that the gene protein was successfully expressed.Immunofluorescence and telomere Fish showed that TPP1-CGFP can be recruited to the telomere region,and plasmid co-transformation can reassemble the split proteins into GFP.Conclusion:The eukaryotic expression vector was successfully constructed and proved that TPP1-CGFP can be recruited to the telomere region,and the split GFP protein can be reassembled by the interaction of connexins and emit green fluorescence.This paper provides a visual cell model for the study of telomerase and TPP1 protein and the interaction of telomeres.