Prokaryotic Expression And Identification And Construction Of The Eukaryotic Expression Vector Of Human MGL

Dendritic cell (DC) is a kind of antigen presenting cell and plays a key role in theimmune responses. DC express a variety of pattern recognition receptors (PRRs), includingToll-like receptors (TLRs) and C-type lectin receptors (CLRs). The TLRs mainly mediatedimmune activation through the interaction with the pathogen.The CLRs tend to mediateimmune tolerance. Recently, an accumulating evidences indicated that macrophage galactosetype C-lectin (MGL)，a member of the CLR family, involved in the immune escape ofpathogens.In this study, four prokaryotic expression vectors were constructed，in order to select thebest condition of MGL expresstion. Secondly, two eukaryotic expression vector wereconstructed for cell transfection in different types of cells.Monocytes were ifrstly separated from human peripheral blood and then induced bycytokines into immature dendritic cells (iDC). Total RNA was extracted from iDC andconverted to cDNA. Then MGL extracellular gene fragment was obtained by PCR. Afterdouble enzyme digestion and ligation, two kinds of prokaryotic expression vectors,pET-28-a(+)-MGL(extracellular) and pGEX-6p-1-MGL(extracellular), were constructed. Theprokaryotic expression vector was transferee! into E. coil. BL21. The protein analysis showedthat almost protein existed in the form of inclusion body. For the purpose of a soluble proetin,two kinds of prokaryotic expression vector pET-28-a(+)-MGL and pGEX-6p-1-MGL wereconstructed with MGL overall gene fragment and were induced in E. coli. The proteinanalysis showed that the interested protein of pGEX-6p-1-MGL were expressed in solubleform. Next, we puriifed the fusion protein from supernatant of GEX-6p-l-MGL through theGST agarose aiffnity chromatography method. On the basis of the prokaryotic expressionvector construction, two types of eukaryotic expression vector pcDNA3.1(+)-MGL andpWPXLd-MGL were constructed. The pcDNA3.1(+)-MGL vector is suitable for transfectionof adherent cells and pWPXLd-MGL vector is ift for transfecting into suspension cells.The construction of MGL prokaryotic expression system provides the method for theconstruction of prokaryotic expression vector of other similar lectins.GST tag of GST-MGLfusion protein can be used to the future research of co-receptor and intracellular signaltransduction proteins interacted with MGL. Eukaryotic expression vector can be used in celltransfection. which is also important for the future research of cell-cell interaction mediatedby MGL and MGL-mediated signal pathway.