A cellular model for the expression of the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin: construction and functional analysis
10.3760/cma.j.issn.0412-4030.2014.07.002
- VernacularTitle:C型凝集素受体树突细胞特异性捕获细胞间黏附分子3非整合素细胞表达模型的构建及功能研究
- Author:
Yu ZHANG
;
Xu YAO
;
Hanyan GU
;
Baoxi WANG
;
Jun LIU
- Publication Type:Journal Article
- Keywords:
Receptors,mitogen;
Recombinant fusion proteins;
Antigens,dermatophagoides;
Cell line,tumor
- From:
Chinese Journal of Dermatology
2014;47(7):457-460
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a cellular model for the expression of the C-type lectin dendritic cellspecific 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.
