摘要:目的构建含复制蛋白A70(RPA70)基因的短发夹RNA(shorthairpinRNA,shRNA)表达质粒,诱导RNA干扰(RNAinterference,RNAi)。方法根据GenBank中RPA70基因的mRNA序列,设计、合成2条反向重复多聚寡核苷酸序列,退火形成双链DNA。利用分子克隆技术,将含RPA70基因的双链DNA与经双酶切后的载体psi—U6-GFP—Neo连接,构建psi—U6-GFP—Neo—shRNA—RPA70重组质粒,通过DNA测序证实表达质粒构建成功。设空白对照组、阴性对照组及实验组,在脂质体2000的介导下转染食管癌TE-1细胞株,荧光显微镜下观察绿色荧光蛋白(GFP)的表达情况。结果DNA测序证实含RPA70基因的shRNA表达质粒构建成功。结论成功地构建了重组质粒pSi—U6-GFP—Neo—shRNA—RPA70,为下一步干扰实验奠定了基础。
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