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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 p100 gene RNA interference plasmid vector and test its inhibitory effect on p100 protein expression in dopaminergic MN9D cell line. Methods: Two RNA interference fragments targeting mouse p100 gene were designed and synthesized. After annealed, pSilencerTM3.1-H1 plasmids were respectively inserted into them and named as Sip100 # 1 and Sip100 # 2 respectively. After identification by restriction analysis and sequencing, The recombinant plasmids and negative control plasmids were respectively transfected into MN9D cells. The expression of p100 protein in cytoplasm of each group was detected by Western Blot. Results: (1) Enzyme digestion and sequencing confirmed that the target oligonucleotide fragment was cloned into pSilencerTM3.1-H1 plasmid. (2) Compared with the control group, the MN9D cells transfected with MN9D cells for 48 h The expression of p100 protein was significantly inhibited in Sip100 # 2 group, in which Sip100 # 1 group decreased by 58%, the difference was statistically significant (P <0.01). CONCLUSION: This study successfully constructed mouse p100 gene-targeted RNAi plasmid vector and can effectively inhibit the expression of p100 protein in MN9D cells, providing a tool for further study of the effect of p100 protein signal pathway on dopaminergic neurons.