S1P1真核表达载体的构建及在HEK293细胞膜上的表达

目的构建含人Ⅰ型1-磷酸鞘氨醇受体(SIPI)-全长cDNA序列及HA和/或EGFP标签的真核表达载体,并观察其在HEK293细胞上的表达。方法合成2条HA寡核苷酸,克隆入S1P1-Myc-EGFP-N1载体。以HA-S1P1-Myc-EGFP-N1为模板,用PCR的方法扩增HA-S1P1并克隆入pcDNA3.1(+)载体。限制性酶切消化、PCR、测序的方法鉴定,载体经Polyfect转染入HEK293细胞。G418筛选出稳定细胞株。用流式细胞仪和激光共聚焦显微镜检测S1P1在HEK293细胞上的表达。结果限制性酶切消化、PCR、测序结果证实载体构建成功。S1P1表达在HEK293稳定细胞株的表面。结论成功构建带有HA和/或EGFP标签的S1P1真核表达载体,表面表达S1P1的HEK293稳定细胞株可作为我们进一步研究的细胞模型。

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.