靶向XT-1基因shRNA真核表达质粒载体的构建及筛选  

Establish and selection of plasmid expressive vector coding shRNA targeting XT-1

作　　者：余资江[1] 余德立[2] 

机构地区：[1]贵阳医学院,贵州贵阳550004 [2]贵阳中医学院第二附属医院

出　　处：《山东医药》2009年第3期19-21,共3页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(30660189)

摘　　要：目的构建靶向木糖转移酶-1(XT-1)的短发卡状小干扰RNA(shRNA)质粒表达载体;为寻找脊髓损伤治疗的新靶点提供工具。方法设计4个小分子短发卡状RNA(siRNA)序列,体外合成DNA模板引物,与Pgenesil-1质粒构建成编码shRNAR的表达载体,进行酶切和测序,再转染体外培养的星形胶质细胞,筛选靶序列。结果构建的质粒表达载体完全符合设计要求,转染成功的细胞在荧光显微镜下显示绿色荧光,G418可以筛选出阳性克隆。结论成功构建了编码XT-1-shRNA的质粒表达载体;该载体为寻找脊髓损伤治疗的新靶点奠定了基础。Objictive To establish and select the plasmid expressive vector ceding short hairpin RNA （shRNA） targeting xylosyhransferase-1 （ XT-1 ） , in order to find a new therapeutic target for the treatment of spinal cord injury. Methods Four sequences of small interference RNA （siRNA） were designed, DNA template primer of XT-1 was synthesized in vitro and the plasmid expressive vectors coding shRNA were established with pGenesil-1 plasmid, then were identified by digestion and tested for the sequence. The astrocyces were transfected with plasmid victor, and then were taken fluorescence photographs and selected by G418. Results The digestion identification of plasmid vectors confirmed that DNA template primer was successfully inserted in the plasmid and the sequence was in conformity with the designed result. After the plasmid vector with green fluorescent protein was transfected to the ceils, it was seen as a green fluorescent wave. The positive clones were also successfully selected. Conclusion Plasmid expressive vectors ceding XT-1 shRNA can be successfully established and capable of stable transfection, and which provided a new therapeutic foundation for the treatment of spinal cord injury.

关 键 词：硫酸软骨素蛋白多糖 木糖转移酶-1 小分子干扰RNA 短发卡状RNA 质粒载体 

分 类 号：R322.81[医药卫生—人体解剖和组织胚胎学]