microRNA-141及HMGB1shRNA构建及干扰效果的验证

发布时间：2018-08-19 20:43

【摘要】：目的：构建有效靶向小鼠HMGB1基因的microRNA-141及HMGB1shRNA干扰载体。 方法：1.由miRBase数据库查找获得microRNA-141成熟序列,设计目的基因片段并人工合成,将microRNA-141序列和载体pYr-miR-30-shRNA经双酶切后连接生成pYr-adv-mmu-miR141重组质粒,用BglⅡ、XhoⅠ双酶切后测序比对；重组质粒转染小鼠肝癌细胞株Hepal-6,采用Q-PCR、Western blot验证干扰效果。2.针对小鼠HMGB1基因序列设计4条HMGB1shRNA干扰序列：pYr-1.1-musHMGB1sh1～4,XhoⅠ酶切后测序比对；重组质粒转染小鼠肝癌细胞株Hepal-6,采用Q-PCR、Western blot验证干扰效果。 结果：1.经酶切凝胶电泳证实microRNA-141序列构建正确,转染microRNA-141的小鼠肝癌细胞株Hepal-6中HMGB1表达量明显低于阴性对照组及未处理组,差异有统计学意义(P0.05)。2.经酶切凝胶电泳证实pYr-1.1-musHMGB1sh1～4序列构建正确,但质粒筛选实验表明pYr-1.1-musHMGB1sh1～4干扰效果与对照组比较没有明显差异(P0.05)。 结论：1. pYr-adv-mmu-miR141序列构建正确,且能成功抑制hepal-6细胞株HMGB1基因的表达。2. pYr-1.1-musHMGB1sh1～4序列构建正确,但不能成功抑制hepal-6细胞株HMGB1基因的表达。
[Abstract]:Objective: to construct microRNA-141 and HMGB1shRNA interference vectors targeting mouse HMGB1 gene. Method 1: 1. The mature sequence of microRNA-141 was obtained from miRBase database. The target gene fragment was designed and synthesized. The microRNA-141 sequence and vector pYr-miR-30-shRNA were ligated into pYr-adv-mmu-miR141 recombinant plasmid after double enzyme digestion. The recombinant plasmid was sequenced and compared with Bgl 鈪，

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