小鼠IDO真核表达载体的构建及其在未成熟树突状.doc

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 安晓静1，钱桂生1，王长征1，夏俊波1，党涛2，廖伟1（第三军医大学新桥医院：1全军呼吸内科研究所；2新桥医院心血管内科，重庆 400037） 基金项目:国家自然科学基金资助项目 Supported by the National Natural Science Foundation of China 作者简介:安晓静(1976 - ) ,女,山东省泰安市人,博士生,医师,主要从事哮喘发病机制方面的研究。电话: ( 023 ) E-mail: anxiaojing@yahoo. cn 通信作者:廖伟,电话: (023)收稿日期: 2008-02-26;修回日期: 2008-3-12 摘要： 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IDO基因全长，DNA重组技术将其定向插入到真核表达载体pEGFP-Ｎ1,, 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IDO基因正确插入pEGFP-Ｎ1载体，与Genank中报道的序列一致pEGFP-Ｎ1-IDO,并转染未成熟树突状细胞,成功地表达目的基因。结论未成熟树突状细胞未成熟树突状细胞 关键词　吲哚胺2,3－双加氧酶；绿色荧光蛋白；真核表达载体中图法分类号： Construction of eukaryotic expression vector containing mouse IDO gene and its expression in immature DCs AN Xiao-jing1, Q IAN Gui-sheng1,WANG Chang-zheng1,XIA Jun-bo1, DANG Tao2,LIAO Wei1(1Institute of Respiratory Diseases, 2Department of Cardiovascular Diseases,Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China) Abstract：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 microscope 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-I