CAJ | 학술논문

目的构建有效靶向小鼠高迁移率族蛋白B1 (HMGB1)基因的短发卡RNA(shRNA)载体.方法针对小鼠HMGB1序列设计4条HMGB1干扰序列的shRNA载体:pYr-1.1-musHMGB1-sh1～4以及与哺乳动物基因组无同源性序列的shRNA作为对照(NC)组,Xho Ⅰ酶切鉴定其序列.然后利用质粒转染小鼠肝癌细胞株Hepal-6,实时荧光定量聚合酶链反应(FQ-PCR)检测干扰效率.结果经酶切凝胶电泳证实shRNA构建正确;质粒筛选结果表明,转染48h后,pYr-1.1-musHMGB1-sh1～4及NC组的荧光表达率为85％、78％、75％、70％、60％;FQ-PCR显示pYr-1.1-musHMGB1-sh1～4组及NC组转染Hepal-6细胞48h后相对表达量分别为0.52±0.05、0.59±0.08、0.61±0.08、0.57±0.21、1.02±0.21.结论成功构建表达靶向HMGB1的4条shRNA重组质粒载体,其中pYr-1.1-musHMGB1-sh1质粒对HMGB1基因的抑制作用最强,为研究HMGB1的功能提供实验基础.
목적 구건유효파향소서고천이솔족단백B1 (HMGB1)기인적단발잡RNA(shRNA)재체.방법 침대소서HMGB1서렬설계4조HMGB1간우서렬적shRNA재체:pYr-1.1-musHMGB1-sh1～4이급여포유동물기인조무동원성서렬적shRNA작위대조(NC)조,Xho Ⅰ매절감정기서렬.연후이용질립전염소서간암세포주Hepal-6,실시형광정량취합매련반응(FQ-PCR)검측간우효솔.결과 경매절응효전영증실shRNA구건정학;질립사선결과표명,전염48h후,pYr-1.1-musHMGB1-sh1～4급NC조적형광표체솔위85％、78％、75％、70％、60％;FQ-PCR현시pYr-1.1-musHMGB1-sh1～4조급NC조전염Hepal-6세포48h후상대표체량분별위0.52±0.05、0.59±0.08、0.61±0.08、0.57±0.21、1.02±0.21.결론 성공구건표체파향HMGB1적4조shRNA중조질립재체,기중pYr-1.1-musHMGB1-sh1질립대HMGB1기인적억제작용최강,위연구HMGB1적공능제공실험기출.
Objective To construct effective short hairpin (shRNA) recombinant plasmids targeting mouse high mobility group box-1 protein (HMGB1) gene.Methods Four pairs of shRNA sequences targeting mouse HMGB1 gene and one pair of negative control shRNA sequence were designed and synthesized.The recombinant plasmids were constructed and identified by Xho Ⅰ restriction enzyme.Then the four plasmids were transfected into mouse hepatoma cell line Hepa1-6 for shRNA screening.HMGB1 knockdown efficiency was evaluated by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) detecting system analysis.Results The four shRNA recombinant plasmids were constructed correctly.The shRNA screening experiment identified that the fluorescence expression rate of pYr-1.1-musHMGB1-sh1-4 and NC is 85％,78％,75％,70％ and 60％ ; FQ-PCR shows relative expression levels of pYr-1.1-musHMGB1-sh1-4 and NC is 0.52 ± 0.05,0.59 ± 0.08,0.61 ± 0.08,0.57 ±0.21 and 1.02 ±0.21 respectively.Conclusion Four recombinant plasmid of HMGB1-targeted shRNA is successfully constructed and screened,in which pYr-1.1-musHMGB1-sh1 exerted the most significant inhibition effect on HMGB1 gene,providing the basis for studying on HMGB1 function.