The Construction Of Eukaryotic Expression Vector And Cell Location Of Human MGL

MGL is a kind of C-type lectin expressed on the surface of immature dendritic cell (iDC) and macrophages with the capacity of specific recognizing N-acetyl galactosamine (GalNAc), such as Tn antigen (Ser/Thr-GalNAc). Although MGL could recoginze its ligands expressed multiple pathogens and some tumor cells, the MGL mediated signaling pathways is still unclear. As we known, iDC for research in lab is obtained only through induction of human peripheral blood monocytes. Because this inducing course is quite difficult, as well as DC could not proliferate in vitro, the establishment of a cell model for study on MGL signaling pathways is improtant.In this study, we fistly constructed MGL eukaryotic expression vector. After that, the transient and stable transfection system was developed respectively. At last, we preliminary study on the location of MGL where in or on cell.pcDNA3.1+-MGL, pcDNA3.1+-MGL-Flag eukaryotic expression vectors for transient transfection system and pWPXLd-MGL, pWPXLd-MGL-Flageukaryotic expression vectors for stable transfection system were contructed successfully. Each series of eukaryotic expression vector included complete MGL gene, MGL△2-8(YENF motif deficient) and MGL2-31(YENF and LL motifs deficient). LipofectamineTM2000and Amaxa electronic transfection were used for transient transfection system contruction, and lentivirus packaging and infection were used for stable transfection system. FACS and Western blot results showed that the transient and stable transfection cell lines were constructed successfully. In research of MGL location, we observed that Flag tag dose not influence MGL expression on the surface of cell. Morever, MGL not only located on cell membrane, but also in cytoplasm, indicating that MGL may rapidly translocation to membrane under specific stimulation. MGL expresstion in cytoplasm and cell surface was increased under the long time stimulation of dexamethasone (Dex), while shot time stimulation the increase took place in cytoplasm only, suggesting that Dex may induce immune tolerance.The novel cell lines for study on MGL mediated signaling pathways was established, which provide important foundation for further reveal MGL physiological functions and MGL mediated celluar signaling pathways.