基础医学与临床 ›› 2013, Vol. 33 ›› Issue (7): 898-903.

• 技术与方法 • 上一篇    下一篇

Neuralized siRNA表达载体的构建及鉴定

朱美意,单莉娅,郑艳,郝慧鑫,黄瑾   

  1. 新疆石河子大学医学院新疆地方与民族高发病教育部重点实验室/生物化学教研室
  • 收稿日期:2013-01-11 修回日期:2013-03-19 出版日期:2013-07-05 发布日期:2013-06-26
  • 通讯作者: 黄瑾 E-mail:huangjin623@163.com
  • 基金资助:
    Neuritin通过 Neuralized 蛋白调节Notch信号通路的机制研究

Construction and identification of siRNA expression vector of neuralized

  • Received:2013-01-11 Revised:2013-03-19 Online:2013-07-05 Published:2013-06-26

摘要: 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 均能影响使定位于细胞膜上的Nneuralized 蛋白的荧光强度。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。

关键词: neuralized, SiRNA干扰

Abstract: Objective To construct Neuralized siRNA vector and to observe the effect of siRNA plasmids on expression of Neuralized in cells. Method Short hairpin oligonucleotide sequence producted by chemical synthesis was first inserted into linearized plasmid. After indentificated by enzymatic digestion and sequencing, Neuralized siRNA plasmids were transfected into HEK293 cells, the expression level and change of location of Neuralized were analysis by Western-Blot and Immunofluorescence. Result Sequencing result from Neuralized siRNA vector showed that 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 displayed that neuralized expression was decreased in cells. Immunofluorescence displayed that the fluorescence of neuralized lacted on the cell membrane was dimmed when the cells were transfected the four kinds of neuralized siRNA plasmids. Conclusion The neuralized siRNA plasmids were successfully constructed and could decrease expression level of neuralized in cells.

Key words: Neuralized, SiRNA-interference