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目的:利用红色荧光蛋白(RFP),建立一种直观、快速筛选有效RNA干扰片段的方法。方法:通过分子克隆技术将核迁移蛋白(NUDC)与RFP构建成融合基因,克隆至真核表达载体pDs中,以实现其融合表达;同时,将人U6启动子及9个NUDC的发夹结构分别克隆至上述同一真核载体中,构建成一系列针对NUDC不同干扰位点的RNA干扰载体,通过采用酶切及DNA序列鉴定,然后转染293T细胞,通过荧光显微镜观察293T细胞的荧光发光强度及荧光细胞数。结果:酶切及测序结果证实质粒为所需的序列;荧光显微镜观察结果显示,shNUDC-A的干扰效果最好。结论:成功构建了含RFP的NUDC真核shRNA干扰载体。
Objective: To establish an intuitive and rapid screening method for effective RNA interference fragments by using red fluorescent protein (RFP). METHODS: NUDC and RFP were constructed into fusion gene by molecular cloning technique and cloned into eukaryotic expression vector pDs to achieve their fusion expression. At the same time, human U6 promoter and hairpin structure of 9 NUDCs Were cloned into the same eukaryotic vector respectively to construct a series of RNA interference vectors targeting different NUDC interference sites. The recombinant plasmid was digested with restriction endonucleases and identified by DNA sequencing. Then 293T cells were transfected with 293T cells. Fluorescence microscopy was used to observe the fluorescence intensity of 293T cells. Fluorescent cell number. Results: The plasmid was confirmed by restriction enzyme digestion and sequencing. Fluorescence microscopy showed that shNUDC-A had the best interference effect. Conclusion: NUDC eukaryotic expression vector containing RFP was successfully constructed.