p100基因RNA干扰质粒载体的构建与鉴定  

Screening and construction of p100-gene RNA interference plasmid vector

作　　者：黄贤贵[1,2] 钮洪艳[2,3] 高秀先[2,4] 袁红花[2] 高殿帅[2] 

机构地区：[1]柳州市人民医院儿科,柳州545006 [2]徐州医学院神经生物学教研室,徐州221002 [3]淮安市第二人民医院,淮安223002 [4]九江市第一人民医院,九江332000

出　　处：《神经解剖学杂志》2014年第4期403-407,共5页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(30870797)

摘　　要：目的:构建p100基因RNA干扰质粒载体,并检测其对多巴胺能的MN9D细胞系中p100蛋白表达的抑制作用。方法:设计并合成2条针对小鼠p100基因的RNA干扰片段,退火后分别连入pSilencerTM3.1-H1质粒并分别命名为Sip100#1、Sip100#2,酶切和测序鉴定正确后,将以上重组质粒及阴性对照质粒分别转染MN9D细胞,运用Western Blot方法检测各组细胞胞浆内p100蛋白的表达水平。结果:(1)酶切和测序结果证实目的寡核苷酸片段已被准确克隆到pSilencerTM3.1-H1质粒;(2)与对照组相比,转染MN9D细胞48 h后Sip100#1组及Sip100#2组p100蛋白的表达水平受到明显抑制,其中Sip100#1组下降58%,差异有统计学意义(P<0.01)。结论:本研究成功构建出小鼠p100基因靶向RNAi质粒载体,并能有效抑制MN9D细胞内p100蛋白的表达,为进一步研究p100蛋白所在信号通路对多巴胺能神经细胞的作用提供了工具。Objective： To construct a p100 gene RNA interference （siRNA） plasmid vector, and determine its inhibitory effect on the expression of p100 protein in the dopaminergic MN9D cells. Methods： Two pairs of double-stranded DNAs encoding siRNA against mice p100 were designed and synthesized chemically. After reannealing, the double-stranded DNAs conjuncted with pSilencer^TM 3. 1-H1 and be named with Sip100#1, Sip100#2, respectively. The positive recombinant was indentified by EcoRI and sequence analysis, then transfected into MN9D cells. The expression level of p100 protein was detected by Western Blot. Results： （1） Restriction enzyme digestion and sequencing analysis results confirmed that the target oligonucleotide fragments have been correctly cloned into plasmid pSilencerTM 3. 1-H1. （2） Compared with the control group, the expression of p100 protein in the MN9D ceils were significantly inhibited after 48 h of transfection of Sip100#1 or Sip100#2. Among them, the Sip100#1 group decreased 58%, the difference was statistically significant （ P 〈 0.01 ）. Conclusion ： The present study successfully construct the RNAi plasmid which targeting to mice p100 gene and effectively inhibited expression of p100 protein in MN9D cells after transfecting of RNAi plasmid, providing the tool for further studying the role of signaling pathway which contains the p100 protein in dopaminergic neuronal cells.

关 键 词：RNA干扰 P100 MN9D细胞系 

分 类 号：R346[医药卫生—基础医学]