摘要
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、Westernblot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 enhanced green fluorescent protein （pEGFP-N1） and expression vector containing its expression in immature mouse IDO gene fused with DCs. Methods The full-length IDO gene was obtained from mouse by RT-PCR and was inserted into eukaryotic expression vector pEG- FP-N1. After identified by restriction digestion and sequencing, the recombinant pEGFP-NI-IDO was transfected into immature DCs by DOTAP liposome. After screened by G418, the green fluorescence protein expression in immature DCs was observed under inverted fluorescence microscope and the mRNA and protein expressions of IDO were respectively detected by RT-PCR and Western blot. Results The full-length sequence of IDO was successfully inserted into the eukaryotic expression vector pEGFP-N1 and confirmed by restriction digestion and sequencing. The recombinant was successfully transfected into immature DCs and the IDO gene was expressed. Conclusion The eukaryotic expression vector pEGFP-NI-IDO was constructed successfully and IDO gene could be expressed efficiently in immature transfected DCs.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2008年第11期1033-1036,共4页
Journal of Third Military Medical University
基金
国家自然科学基金(30500231)~~
