中国生物工程杂志

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-v5tag vector. After bacterial liquid PCR, vector enzyme digestion and sequencing verification, it was transfected into 293T 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 293T 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.