發(fā)布時間：2018-08-19 20:43

【摘要】：目的：構建有效靶向小鼠HMGB1基因的microRNA-141及HMGB1shRNA干擾載體。 方法：1.由miRBase數據庫查找獲得microRNA-141成熟序列,設計目的基因片段并人工合成,將microRNA-141序列和載體pYr-miR-30-shRNA經雙酶切后連接生成pYr-adv-mmu-miR141重組質粒,用BglⅡ、XhoⅠ雙酶切后測序比對；重組質粒轉染小鼠肝癌細胞株Hepal-6,采用Q-PCR、Western blot驗證干擾效果。2.針對小鼠HMGB1基因序列設計4條HMGB1shRNA干擾序列：pYr-1.1-musHMGB1sh1～4,XhoⅠ酶切后測序比對；重組質粒轉染小鼠肝癌細胞株Hepal-6,采用Q-PCR、Western blot驗證干擾效果。 結果：1.經酶切凝膠電泳證實microRNA-141序列構建正確,轉染microRNA-141的小鼠肝癌細胞株Hepal-6中HMGB1表達量明顯低于陰性對照組及未處理組,差異有統計學意義(P0.05)。2.經酶切凝膠電泳證實pYr-1.1-musHMGB1sh1～4序列構建正確,但質粒篩選實驗表明pYr-1.1-musHMGB1sh1～4干擾效果與對照組比較沒有明顯差異(P0.05)。 結論：1. pYr-adv-mmu-miR141序列構建正確,且能成功抑制hepal-6細胞株HMGB1基因的表達。2. pYr-1.1-musHMGB1sh1～4序列構建正確,但不能成功抑制hepal-6細胞株HMGB1基因的表達。
[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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