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

目的 构建小鼠pEGFP-N1-IDO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, 用倒置荧光显微镜观察转染的未成熟树突状细胞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 .

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

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 identified by restriction digestion and sequencing,the recombinant pEGFP-N1-IDO was transfected into immature DCs by DOTAP liposome.After screened by G418,the green fluorescence protein expression in immature ...