Construction of eukaryotic expression vector harboring human DC-SIGN gene and identification of expressed protein in K-562 cells

Objective To construct the eukaryotic expression vector coding human DC-SIGN gene, and to express DC-SIGN protein in K-562 cells for investigating the interactions of dendritic cells (DC) with HCV. Methods Peripheral blood monocytes (PBMC) isolated from blood of healthy individuals were stimulated by GM-CSF and IL-4 and differentiated into immature DCs. The whole coding region of DC-SIGN was initially obtained from those differentiated DCs by RT-PCR using designed primers. The resulting fragment was digested with EcoRⅤ and HindⅢ and gel purified and ligated into EcoRⅤ- and HindⅢ-digested pCDNA3.1- to generate pCDNA3.1-DC-SIGN. Transfection of pCDNA3.1-DC-SIGN in K562 cells was accomplished using Lipofectamine2000. The expression of DC-SIGN was verified by direct immunofluorescence using the PE-conjugated mouse monoclonal anti-human DC-SIGN. Results human DC-SIGN gene was successfully constructed into eukaryotic expression vector and transfected into K-562 cells. The level of DC-SIGN's expression on transfected cells was high. Conclusion DC-SIGN could expressed on K-562 cells at high level. The results would contribute to further studies of effects of DC-SIGN on HCV infection.