重组表达载体IDO/pEGFP-C1的构建  

Construction of recombinant expression vector of IDO/pEGFP-C1

作　　者：陈琬玲 李臣 范立 师娟 甘泉 李永芬 苏瑾 蒋璧谦 

机构地区：[1]武警云南总队医院肿瘤中心,云南昆明650111

出　　处：《武警后勤学院学报（医学版）》2015年第2期85-88,F0002,共5页Journal of Logistics University of PAP（Medical Sciences）

基　　金：云南省科技厅昆明医学院应用基础研究联合专项资金项目(2008CD033)

摘　　要：【目的】构建表达IDO重组表达载体IDO/p EGFP-C1。【方法】RT-PCR扩增IDO c DNA,连接于表达载体p EGFP-C1,酶切鉴定及序列分析证实后将载体转染至TC-1细胞,RT-PCR和Western blotting实验观察IDOm RNA和蛋白表达。【结果】酶切鉴定及序列分析证实IDO成功连接于表达载体p EGFP-C1上,转染至TC-1细胞后可见IDOm RNA和蛋白表达。【结论】IDO重组表达载体IDO/p EGFP-C1构建成功。[Objective]To construct the expression vector ofIDO （IDO/pEGFP-C1）. [Methods]IDO eDNA was amplified by RT-PCR from RAW 264.7 cells stimulated by recombinant mouse interferon-＇y 200 U/ml .The product of PCR was excised with BgII] and Kpnl and site in the pcDNA3.1/Zeo（＋） vector which was excised with BgIII and Kpnl. The expression vector of IDO （IDO/pEGFP-C 1） was confirmed by sequence analysis. The IDO/pEGFP-C1 vector was transfected into TC-1 cell using lipofectamine 2 000. Stably transfected lines were selected in 400 U/ml Kan. RT-PCR and Western blottong were used to detected the IDO mRNA and protein expression in IDO/pEGFP-C 1 TC-cells. [ Results ] The expression vector of IDO （IDO/pEGFP-C 1） excised with BgⅢ and Kpni and the 1.2 kb band was observed. Sequence was confirmed by sequence analysis. The IDO cDNA band in 233 bp and IDO protein band in 45 kd were detected by RT-PCR and Western blotting. [ Conclusions ]The expression vector of IDO （IDO/pEGFP-C 1） was constructed successfully.

关 键 词：吲哚胺2 3二氧化酶 表达载体 基因重组 

分 类 号：R393[医药卫生—基础医学]