摘要目的原核表达小鼠Flt3配体,分析其在不同亚型DCs体外诱导中的应用潜能,为不同亚型DCs的深入研究提供实验资料。方法 PCR扩增flt3l基因,构建pCold-flt3l质粒,转化大肠埃希菌BL21(DE3)诱导表达并纯化。重组蛋白进行SDS-PAGE和Western blot分析,并用于小鼠骨髓源DCs的诱导制备,流式细胞术分析不同DC亚型相关分子标志以及CD40、CD80、CD86和MHCⅡ的表达,同时ELISA测定IL-12和IFN-α的表达。结果 SDS-PAGE结果表明目的蛋白成功表达,纯化后纯度达93.3%,Western blot结果表明其具备良好的免疫反应性。同时,该蛋白能诱导小鼠骨髓细胞分化为较高比例(约60%)的DCs,且包含cDCs( CD11c+CD45RA-)和pDCs( CD11c+CD45RA+)亚型；LPS刺激后2种DC亚型均上调CD40、CD80、CD86和MHCⅡ的表达,且分泌较高水平的 IL-12和IFN-α。结论本研究成功获得重组小鼠Flt3配体蛋白,可用于制备具有良好生物学功能的cDCs和pDCs亚型,为下一步研究提供基础材料。

abstracts<br> 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 expres-sion 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 gen-erated with the recombinant FL protein. Flow cytometry ( FCM) analysis was performed to detect the pheno-typic 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&nbsp;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-clas-sic 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 en-hanced secretion of IL-12 and IFN-α in response to LPS stimulation. Conclusion In this study, we suc-cessfully expressed the murine FL protein which could be used for the preparation of different DC subsets.