Construction of the Eukaryotic Expressing Vectors of S1P1 and its Expression on the Surface of HEK293 Cells

Objective To construct the eukaryotic expression vectors containing the coding region of human full length sphingosine 1-phosphate receptor typeⅠ(S1P1) gene with hemagglutinin(HA) and/or EGFP tag,and to detect its expression in HEK293 cells.Methods Two DNA oligonucleotides encoding HA were synthesized,and cloned into S1P1-Myc-EGFP-N1 vector.HA-S1P1 was amplified from HA-S1P1-Myc-EGFP-N1 plasmid by PCR and cloned into pcDNA3.1(+) vector.The recombinant vectors were confirmed by restriction enzyme digestion,PCR and sequencing,then they were transfected into HEK293 cells by Polyfect.The transfected HEK293 cells were selected with G418.Flow cytometry and confocal laser scanning microscopy were used to detect the S1P1 protein expression in HEK293 cells.Results The results of the restriction enzyme digestion,PCR and sequencing demonstrated the vectors were successfully constructed.S1P1 protein was expressed on the stably transfected HEK293 cell surface.Conclusion Eukaryotic expression vectors containing the S1P1 gene with HA and/or EGFP tag were successfully constructed and S1P1 was expressed on the surface of stably transfected HEK293 cells,which was be used as cell models for further studies.