【摘要】：目的：構(gòu)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(shù)據(jù)庫(kù)查找獲得microRNA-141成熟序列,設(shè)計(jì)目的基因片段并人工合成,將microRNA-141序列和載體pYr-miR-30-shRNA經(jīng)雙酶切后連接生成pYr-adv-mmu-miR141重組質(zhì)粒,用BglⅡ、XhoⅠ雙酶切后測(cè)序比對(duì)；重組質(zhì)粒轉(zhuǎn)染小鼠肝癌細(xì)胞株Hepal-6,采用Q-PCR、Western blot驗(yàn)證干擾效果。2.針對(duì)小鼠HMGB1基因序列設(shè)計(jì)4條HMGB1shRNA干擾序列：pYr-1.1-musHMGB1sh1～4,XhoⅠ酶切后測(cè)序比對(duì)；重組質(zhì)粒轉(zhuǎn)染小鼠肝癌細(xì)胞株Hepal-6,采用Q-PCR、Western blot驗(yàn)證干擾效果。 結(jié)果：1.經(jīng)酶切凝膠電泳證實(shí)microRNA-141序列構(gòu)建正確,轉(zhuǎn)染microRNA-141的小鼠肝癌細(xì)胞株Hepal-6中HMGB1表達(dá)量明顯低于陰性對(duì)照組及未處理組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。2.經(jīng)酶切凝膠電泳證實(shí)pYr-1.1-musHMGB1sh1～4序列構(gòu)建正確,但質(zhì)粒篩選實(shí)驗(yàn)表明pYr-1.1-musHMGB1sh1～4干擾效果與對(duì)照組比較沒(méi)有明顯差異(P0.05)。 結(jié)論：1. pYr-adv-mmu-miR141序列構(gòu)建正確,且能成功抑制hepal-6細(xì)胞株HMGB1基因的表達(dá)。2. pYr-1.1-musHMGB1sh1～4序列構(gòu)建正確,但不能成功抑制hepal-6細(xì)胞株HMGB1基因的表達(dá)。
[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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