摘要
目的构建含红色荧光蛋白DsRed2的RNA干扰质粒载体pDsRed2-SilencerU6,便于利用荧光显微镜、FACS等实验技术开展RNA干扰研究。方法质粒pDsRed2-N1中DsRed2蛋白的编码基因BbsⅠ酶切位点无意突变后,破坏pDsRed2-N1多克隆位点,PCR扩增CMV启动子、DsRed2蛋白基因片段并将其克隆至RNA干扰载体pSilencerU6,得到pDsRed2-SilencerU6质粒,利用脂质体转染技术将其转染HEK293T细胞,荧光显微镜、FACS检测转染效率。结果DsRed2蛋白编码基因序列BbsⅠ点突变正确,成功构建pDsRed2-SilencerU6载体,其在HEK293T细胞中可显著表达红色荧光蛋白,利用脂质体转染技术,转染效率达70.61%。结论成功构建含红色荧光蛋白DsRed2的RNA干扰载体pDsRed2-SilencerU6并在哺乳动物细胞中表达,为今后开展RNA干扰研究提供了基础。
Objective To construct a pDsRed2-SilencerU6 vector with red fluorescent protein DsRed2 which can be used in RNA interference method to express short hairpin RNA （shRNA）. Methods A point mutation at Bbsl recognized sequence site was made on the DNA sequence encoding DsRed2 protein of pDsRed2-N1 vector, and the multiple cloning sites （MCS） of pDsRed2-N1 vector was destroyed. Then the CMV promoter and DsRed2 protein sequence in modified pDsRed2-N1 vector was amplified by PCR and subcloned into mouse U6 promoter based shRNA expression vector pSilencerU6. The recombinant vector pDsRed2-SilencerU6 was transfected into HEK 293T cells by lipofectamine method. The DsRed2 protein expression in HEK 293T cells was detected by fluorescent microscope and flow cytometry （FACS）. Results Bbs Ⅰ cutting site in DsRed2 protein was successfully removed and the construction of pDsRed2-SilencerU6 vector was carried and successfully. The recombinant vector was expressed in HEK293T cells, the transfection efficiency was up to 70.6196 by FACS analysis. Conclusion The recombinant vector pDsRed2-SilencerU6 can be used in RNA interferencebased experiments and is convenient for studying in gene function and related research.
出处
《同济大学学报（医学版）》
CAS
2008年第1期17-20,共4页
Journal of Tongji University(Medical Science)
基金
国家自然科学基金资助项目(30570690)
