摘要
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。
AIM：Used RFP gene to construct a RNA interference vector for convenience to obtain the good effective hairpins sequence.METHODS：NUDC and RFP genes were cloned into pDs vector separately,resulting in pDs-NUDC-RFP.as above,human U6 promoter and 9 hairpins sequence of NUDC were cloned into the pDs-NUDC-RFP vector separately.The RNA interfererence vectors target to 9 points of NUDC were constructed.Construct-ed recombinant vectors and then were identified by restrictive digestion and DNA sequencing.293T cells were transfecte with the recombinant DNA samples by the liposome complex method,and then fluorescence photographs were taken.RESULTS：Enzyme digestion and DNA sequencing showed that the target gene and shRNA fragments were correctly inserted in pDs vector.fluorescence photographs showed that shNUDC-A is the best effective fragment.CONCLUSION：The NUDC gene targeted shRNA and its vector are successfully constructed.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2011年第7期740-743,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
教育部科学技术研究重点项目(210156)
广东省卫生厅青年基金(B2010235)
广东医学院博士启动基金(XB1007)
广东省大学生创新实验项目(KY1003)
