构建真核表达质粒载体pIRES2-EGFP-FLT3及其在脐血CD34^+细胞中的表达  

Construction of eukaryotic expression plasmid vector pIRES2-EGFP-FLT3 and its expression in CD34^+ cells derived from human umbilical blood

作　　者：张勇[1] 张林生[1] 郭朝华[2] 张红宾[1] 裴莉[1] 钟大平[1] 

机构地区：[1]解放军第三军医大学西南医院血液科,重庆市400038 [2]解放军第三军医大学全军复合伤研究所,重庆市400038

出　　处：《中国临床康复》2005年第42期4-5,25,i0001,共4页Chinese Journal of Clinical Rehabilitation

摘　　要：目的:构建真核表达的双顺反子质粒载体pIRES2-EGFP-FLT3配体,为研究Flt3配体对急性放射复合伤造血功能恢复作用提供实验物质基础。方法:实验于2003-05/09在第三军医大学劳动卫生教研室完成。先用SalⅠ和BglⅡ双酶切pUM VC3-hFLT3配体,得到全长的人FLT3配体序列(0.8kb),两端抹平后克隆至pIRES2-EGFP载体的BglⅡ位点。然后用NheⅠ和XhoⅠ酶切鉴定;最后用脂质体转染的方法将重组质粒转入脐血CD34+细胞中,经过G418筛选,在荧光显微镜下观察其表达。结果:①构建了双顺反子真核表达载体pIRES2-EGFP-FLT3。②重组质粒pIRES2-EGFP-FLT3能在脐血CD34+细胞中的表达,凝胶电泳和测序结果证明将FLT3cDNA亚克隆入pIRES2-EGFP内,荧光显微镜下可见脐血CD34+细胞胞体发出绿色荧光。③转染效率为10.3%。结论:①携带标记基因的双顺反子质粒载体pIRES2-EGFP-FLT3能稳定地转入真核细胞表达载体。②该质粒转染到分离纯化的脐血CD34+细胞中,并获得阳性转染细胞的克隆。AIM： To construct a eukaryotic expression bicistron plasmid vector plRES2-EGFP-FLT3 in order to investigate the role of FLt3 in the hemopoiesis and immune function during the recovery of acute radiation combined injury. METHODS： The experiment was completed in the Department of Occupational Hygiene of the Third Military Medical University of Chinese PLA between May and September 2003. After double digestion of pUMVC3-hFLT3 with Sal I and gl II, the product sequence （0.8 kb）, was subcloned into the Bgl II site of plasmid plRES2-EGFP. After identification with Nhe I and Xho I digestion, the recombinant was transfected into CD34^＋ ceils derived from human umbilical blood with liposome transfection technique, and screened with G418 followed by Fh3 fluorescence microscopy. RESULTS： ①A eukaryotic expression bicistron plasmid vector plRES2-EGFP-FLT3 was constructed.② The recombined plasmid plRES2-EGFP-FLT3 could be expressed in human umbilical blood CD34^＋ cells, the cDNA were confirmed by restriction enzyme digestion, sequencing. Green fluorescence was observed after its transfection into CD34^＋ cells. ③ Transfection efficiency was 10.3%. CONCLUSION： ① Plasmids vector plRES2-EGFP-FLT3 with marker gene could be stably transfected and expressed in eukaryotic ceils. ② The plasmid was transfected into isolated and purified CD34^＋ cells in umbilical blood, and the clone of positively transfeeted cells also can be obtained.

关 键 词：抗原 CD34 质粒 转染 

分 类 号：R394.2[医药卫生—医学遗传学]