Cloning of Human DC-SIGN Gene and Construction of Its Transgenic Cells

Objective To clone human DC-SIGN gene and construct recombinant retrovirus vector carrying the target gene which can be expressed stably in mammal cell line L929.Methods Human DC-SIGN gene was amplified by RT-PCR using the mRNA of dendritic cells derived from human peripheral blood monocytes as template and then confirmed by DNA-sequence analysis.Digested with the restriction endonucleases EcoRI and BamHI,the human DC-SIGN gene was inserted into retrovirus vector pGEZ-Term.The recombinant retrovirus vector together with its two helper virus vectors was cotransfected into the package cell 293T in the context of LipfectAMINE.Then the supernatant of 293T was used to infect L929 cells.After 72 hours,L929 cell line stably expressing human DC-SIGN protein was selected in the presence of Zeocin(500μg/ml).Results The full-length of human DC-SIGN gene was cloned and the recombinant retrovirus vector was constructed,The L929 cell line expressing human DC-SIGN was successfully transfected and selected.Results of RT-PCR and flow cytometry indicated that L929 transgenic cells can stably express human DC-SIGN protein on the membrane of cells.Conclusion Cloning of human DC-SIGN gene and construction of the recombinant retrovirus vector and L929 transgenic cell line stably expressing DC-SIGN protein can contribute to further biological function research and monoclonal antibody preparation.