2016•Unpublished venueRequires access
Expression of murine Flt3 ligand and its application in the in vitro preparation of different dendritic cell subsets
Chuang Meng, Xiaoyan Wang, Zhengzhong Xu, Jiaying Liu, Maozhi Hu, Zhiming Pan, Xiang Chen, Xinan Jiao
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Abstract
Objective
To express the murine Flt3 ligand (FL) protein in a prokaryotic expression system and to evaluate its application in the in vitro preparation of different dendritic cell (DC) subsets.
Methods
A fragment of flt3l gene was amplified by PCR and then used to construct the recombinant expression plasmid pCold-flt3l. The transformed E. coli BL21(DE3) carrying expression plasmid were induced by IPTG to express FL protein. The expressed FL fusion protein was purified by using His bind purification kit. SDS-PAGE and Western blot assay were performed for further analysis. Bone marrow derived-DCs were generated with the recombinant FL protein. Flow cytometry (FCM) analysis was performed to detect the phenotypic markers of different DC subsets and the expression of CD40, CD80, CD86 and MHCⅡ molecules. ELISA was used for the quantitative analysis of IL-12 and IFN-α.
Results
The fusion protein was expressed and purified successfully with a purity of 93.3% as indicated by SDS-PAGE and Western blot assay. FCM analysis showed that the FL protein efficiently induced the differentiation of bone marrow cells into DCs with a CD11c positive rate of more than 60%. Two DCs subsets were identified including CD11c+ CD45RA- classic DCs (CD11c+ CD45RA- cDCs) and CD11c+ CD45RA+ plasmacytoid DCs (CD11c+ CD45RA+ pDCs). Both of the two DCs subsets showed up-regulated expression of CD40, CD80, CD86 and MHCⅡ and enhanced secretion of IL-12 and IFN-α in response to LPS stimulation.
Conclusion
In this study, we successfully expressed the murine FL protein which could be used for the preparation of different DC subsets.
Key words:
Dendritic cells; Flt3 ligand; Subset; Activation and maturation