人SET基因RNA干扰重组质粒载体的构建及其在肝细胞中的表达  

Construction of human SET gene RNA interference recombinant plasmid vector and its expression in L-02 liver cells

作　　者：黄新凤[1] 刘建军[1] 蒋英芝[1] 席仁荣[1] 杨亮[1] 

机构地区：[1]深圳市疾病预防控制中心毒理检验科,广东深圳518020

出　　处：《热带医学杂志》2011年第3期291-294,共4页Journal of Tropical Medicine

基　　金：国家自然科学基金(30972454);深圳市科技计划重点项目(200801010)

摘　　要：目的建立真核质粒载体介导的人SET基因RNA干扰表达体系,并检测其在肝细胞中对SET蛋白表达的影响,为研究SET蛋白在三氯乙烯致肝细胞毒性的作用打下基础。方法设计并合成2对人SET基因特异性短发夹RNA(short hair-pin RNA,shRNA)寡核苷酸链,退火形成双链DNA后插入到真核表达质粒psiRNA-hH1 neo中产生shRNA重组质粒载体,经酶切和测序鉴定成功后,转染至L-02肝细胞中,48h后收集细胞提取总蛋白,Western blotting检测干扰后SET蛋白的表达。结果 shRNA重组质粒的酶切和测序鉴定均正确,Western blotting结果显示重组质粒psiRNA1组的干扰效果较好,对SET蛋白表达的抑制率达60%左右,与正常对照组相比,差异具有统计学意义(P<0.05)。结论成功构建针对人SET基因的RNA干扰真核质粒载体,其能有效地抑制肝细胞内SET蛋白的表达,为深入研究SET蛋白在三氯乙烯致肝细胞毒性中的作用奠定了基础。Objective To construct human patient SE transloction（SET） gene RNA interference recombinant plasmid vector and investigate SET protein＇s expression in the liver cells.The aim of this study was to lay a good basis for the further study of effect of SET protein on trichloroethylene-induced liver cells.Methods The 2 pairs of short hair-pin RNA oligonucleotides targeting human SET gene were designed and synthesized.After annealing,these double-strand DNA were cloned to eukaryotic expression plasmid Vector psiRNA-hH1 neo to form the shRNA recombinant plasmid vector,and they were confirmed to be correctly constructed by digestion and sequencing,and then were used to transfect into L-02 liver cells.48 h after the transfection,the liver cells were harvested,and the expression of SET protein in the liver cells after RNAi was detected by western blotting.Results The digestion and sequencing results confirmed that the recombinant plasmid vectors were correctly constructed,and Western Blotting results showed that transfection of recombinant plasmid psiRNA1 significantly down-regulated SET protein expression in L-02 liver cells,with the inhibition ratio of approximately 60%,P〈0.05,which showed significant difference from the normal control group.Conclusion The human SET gene RNA interference recombinant plasmid vector was successfully constructed,which inhibited SET protein expression effectively,indicating that it laid the groundwork for the study of investigating the role of SET protein in the process of trichloroethylene-induced liver cells.

关 键 词：RNA干扰 SET基因 真核质粒载体 WESTERN BLOTTING 

分 类 号：Q522.2[生物学—生物化学]