Abstract:

Zhang Xiaoning*, Zhang Yu, Luo Yang, Li Wei, Yao Xu. *Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding author: Yao Xu, Email: drYao_xu@126.com 【Abstract】 Objective To establish a cell model expressing the Langerhans cell-specific C type lectin receptor Langerin in vitro. Methods The cDNA of Langerin was obtained by PCR and cloned into a eukaryotic green fluorescent protein （EGFP） expression vector pEGFP-C1 with EGFP located in the N terminal region of the Langerin gene. Then, the recombinant plasmid was transfected into a human embryonic kidney carcinoma cell line HEK293T. Subsequently, laser confocal microscopy was performed to observe the expression of EGFP-Langerin fusion protein, and flow cytometry to measure the expression of Langerin. Laser confocal microscopy was also conducted to visualize the recognition and endocytosis of dust mite antigen （nDer p 2） by Langerin. Results PCR and Western blot confirmed the successful transfection of HEK293T cells with the recombinant plasmid as well as the expression of Langerin in the transfected cells. As flow cytometry revealed, the expression level of Langerin in transfected HEK293T cells was increased by 43% compared with untransfected cells. Laser confocal microscopy showed that green fluorescein-labeled Langerin was successfully expressed, and could bind to and endocytose the red fluorescein-labeled antigen nDer p 2. Conclusions The fusion protein EGFP-Langerin expressed in HEK293T cells showed the distribution characteristic of cell surface receptors, and could bind to and endocytose allergens.