Construction of eukaryotic expression vector of SYAP1 and intracellular location

Aim:To construct eukaryotic expression vector pcDNA3. 1(-)-Myc-SYAP1. Methods:SYAP1 sequence was synthesized,joined with Myc-tag sequence and EcoR Ⅰ,Hind Ⅲ restriction sites,and then connected with linearized pcDNA3. 1(-). After the identification by the results of colony PCR,restriction analysis and sequencing,the plasmid was transfected into HEK293 cells by lipid transfection,Western blot and indirect immunofluorescence were applied to observe the expression level and position of SYAP1 protein in cells. Results:The results of colony PCR,sequencing and restriction enzyme digestion showed that the target nucleotide sequences were successfully inserted into the expected sites of the vector. Western blot showed that SYAP1 protein was successfully expressed in transfected HEK293 cells,and lind indirect immunofluorescence result showed that SYAP1 protein located in cytoplasm. Conclusion:Recombinant vector of pcDNA3. 1(-)-Myc-SYAP1 has been successfully constructed.