目的 构建Neuralized siRNA干扰载体并观察其对细胞中Neuralized表达水平的影响.方法 化学合成靶向Neuralized的短发夹寡核苷酸序列,与线性化pGPU6/Neo质粒退火连接.酶切后测序鉴定正确,转染入HEK293细胞.利用Western blot和间接免疫荧光技术分析Neuralized siRNA载体对细胞中Neuralized表达及其定位的影响.结果 Neuralized siRNA干扰载体经酶切及测序分析表明,目的核苷酸序列成功插入预计位点且序列正确；Neuralized siRNA干扰载体转染HEK293细胞后,Western blot检测显示,Neuralized siRNA干扰载体明显降低细胞中Neuralized蛋白表达；间接免疫荧光显示,转染4种Neuralized siRNA均能使定位于细胞膜上的Nneuralized蛋白的荧光强度变暗.结论 Neuralized siRNA干扰载体成功构建,且能明显抑制细胞中Neuralized的表达.%Objective To constructa neuralized siRNA vector and to observe the effect of siRNA plasmids on expression of neuralized in cells.Methods Short hairpin oligonucleotide sequence producted by chemical synthesis was inserted into linearized plasmid.After indentification by enzymatic digestion and sequencing,neuralized siRNA plasmids were transfected into HEK293 cells,the expression level and change of location of neuralized were analyzed by Western blot and immunofluorescence.Results The target nucleotide sequences were successfully inserted into the expected sites of vectors and sequences were correct.After the plasmids were transfected HEK293 cells,Western blot proved that neuralized expression was decreased in cells.Immunofluorescence showed that the fluorescence of neuralized lacted on the cell membrane was dimmed when the cells were transfected by four kinds of neuralized siRNA plasmids.Conclusions The neuralized siRNA plasmids were successfully constructed and may inhibit expression of neuralized in cells.
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