小鼠IDO真核表达载体的构建及其在未成熟树突状细胞中的表达

Construction of eukaryotic expression vector containing mouse IDO gene and its expression in immature DCs

目的构建小鼠pEGFP-N1-IDO基因真核表达载体并观察其在未成熟树突状细胞中的表达。方法利用RT-PCR方法扩增小鼠IDO基因全长,利用DNA重组技术将其定向插入到真核表达载体pEGFP-N1,经酶切和测序鉴定后,用DOTAP脂质体转染法转染未成熟树突状细胞,通过G418筛选,转染的未成熟树突状细胞,用倒置荧光显微镜观察绿色荧光蛋白的表达,用RT-PCR、Western-blot检测IDO的表达。结果小鼠全长IDO基因序列正确插入pEGFP-N1载体,与GenBank中报道的序列一致,成功构建了pEGFP-N1-IDO真核表达载体,并转染未成熟树突状细胞,成功地表达目的基因。结论真核表达载体成功构建和转染未成熟树突状细胞,并证明能有效表达于未成熟树突状细胞中。

Objective To construct a eukaryotic expression vector containing mouse IDO gene fused with enhanced green fluorescent protein（pEGFP-N1） and its expression in immature DCs.Methods The full-length IDO gene was obtained from mouse by RT-PCR and was inserted into eukaryotic expression vector pEGFP-N1,after the identification by digestion and sequencing on the recombinant eukaryotic expression vector pEGFP-N1-IDO,the recombinant was transfected into immature DCs by DOTAP liposome.After screening culture by G418,immature DCs was transfected.The green fluorescence protein expression was confirmed by inverted fluorescence microscopy and the transcription and expression of IDO were identified by RT-PCR,Western blotting.Results The full-length coding sequence of IDO was inserted into eukaryotic expression vector pEGFP-N1,IDO gene was successfully amplified and identified by DNA sequencing.The eukaryotic expression vector pEGFP-N1-IDO was constructed and transfected into immature DCs successfully.The IDO gene was expressed successfully.Conclusion The construction of the eukaryotic expression vector pEGFP-N1-IDO,IDO gene could be expressed efficiently in immature DCs transfected by pEGFP-N1-IDO.