Abstract: Zhang Yu*, Yao Xu, Gu Hanyan, Wang Baoxi, Liu Jun. *Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding authors: Yao Xu, Email: dryao_xu@126.com; Liu Jun, Email: drliu_jun@126.com 【Abstract】 Objective To establish a cellular model for the expression of the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin （DC-SIGN）, and to provide a basis for the functional analysis of DC-SIGN. Methods The cDNA of DC-SIGN was obtained via PCR, and cloned into the eukaryotic expression vector porcine cytomegalovirus-enhanced green fluorescent protein （PCMV-EGFP） with EGFP at the N terminal of DC-SIGN. Then, the recombinant PCMV-EGFP-DC-SIGN plasmid was transfected into HEK293T cells followed by the detection of DC-SIGN expression using PCR, Western blot and flow cytometry. Confocal microscopy was performed to localize the expression of DC-SIGN-EGFP and visualize the recognization and internalization of the Derp2 allergen by DC-SIGN. Results The recombinant fluorescent fusion protein-expressing plasmid was successfully constructed. Both PCR and Western blot confirmed the expression of DC-SIGN. Flow cytometry showed that the expression of DC-SIGN was increased by approximately 50% in HEK293T cells transfected with the recombinant expression plasmid compared with those untransfected. As confocal microscopy showed, the green fluorescence-labelled DC-SIGN was located on the cell membrane, which could bind to the red fluorescence-labelled antigen Derp2 and internalize it into the cells. Conclusions The recombinant DC-SIGN-EGFP fusion protein is characteristically located on the surface of 293T cells, which can be recognized by the DC-SIGN-specific antibody and is capable of internalizing the allergen Derp2, and may serve as an ideal cell model for further studies on molecular function of DC-SIGN.

Key words: Receptors, mitogen, Recombinant fusion proteins, Antigens, dermatophagoides, Cell line, tumor