小鼠Flt3配体的原核表达及其在不同亚型树突状细胞制备中的应用

Expression of murine Flt3 ligand and its application in the in vitro preparation of different dendritic cell subsets

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。

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 offlt31 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 MHC Ⅱ molecules. ELISA was used for the quantitative analysis of IL-12 and IFN-a. Results The fusion protein was expressed and purified successfully with a purity of 93.3% as indicated by SDS-PAGE and Western blot assay. FCManalysis showed that the FL protein efficiently induced the differentiation of bone marrow ceils into DCs with a CD11 c positive rate of more than 60%. Two DCs subsets were identified including CD11 c＋CD45RA- clas- sic DCs （ CD11 c＋ CD45RA- cDCs） and CD11 c＋ CD45RA＋ plasmacytoid DCs （ CD11 c＋ CD45RA＋ pDCs ）. Both of the two DCs subsets showed up-regulated expression of CD40, CD80, CD86 and MHC Ⅱ and en- hanced secretion of IL-12 and IFN-a 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.