靶向人CHOP基因shRNA真核表达载体的构建

发布时间：2019-07-18 21:41

【摘要】：[目的]构建靶向干扰内质网应激标志性因子CHOP的shRNA真核表达载体,并检测其对CHOP的干扰效率。[方法]查询Gen Bank数据库,获取人源CHOP基因mRNA的序列,按照小干扰RNA(siRNA)靶序列的设计原则,设计并构建靶向CHOP基因mRNA的4个特异性shRNA真核表达载体(shRNA-1、shRNA-2、shRNA-3、shRNA-4)和1个无同源性的阴性对照载体(shRNA-NC),经PCR和测序鉴定确认shRNA载体构建成功后,脂质体转染人正常肝细胞系(L-02),Western Blot法检测CHOP蛋白的表达,筛选出干扰效果最好的表达载体。[结果]PCR和测序结果显示,5个shRNA表达载体均构建成功。Western Blot结果显示,0.06 g/L衣霉素损伤24h后,与内质网应激模型组相比,shRNA-1、shRNA-2、shRNA-3、shRNA-4组的CHOP蛋白表达水平均明显降低(P0.01),其中shRNA-1和shRNA-4组CHOP干扰效果最明显。[结论]构建了并成功筛选出靶向干扰CHOP基因的真核表达载体,为深入研究CHOP介导内质网应激所致细胞凋亡的信号通路奠定了实验基础。
[Abstract]:[objective] to construct shRNA eukaryotic expression vector targeting endoplasmic reticulum stress marker CHOP and detect its interference efficiency to CHOP. [methods] the Gen Bank database was queried to obtain the sequence of human CHOP gene mRNA. According to the design principle of small interference RNA (siRNA) target sequence, four specific shRNA eukaryotic expression vectors (shRNA-1,shRNA-2,shRNA-3,shRNA-4) and one negative control vector (shRNA-NC) targeting CHOP gene mRNA were designed and constructed. The shRNA vector was successfully constructed by PCR and sequencing. The expression of CHOP protein was detected by Liposome transfection into human normal liver cell line (L 鈮，

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