人分化抑制蛋白1慢病毒干扰载体的构建、筛选及其沉默效应的鉴定

Construction, Screening, and Verifi cation of The Silencing Effects for Human Interference Lentiviral Vector of Inhibitor of Differentiation-1

目的探索以小干扰RNA(si RNA)慢病毒干扰载体组建的慢病毒对人Hep G2细胞分化抑制蛋白1(Id1)基因的沉默效应。方法构建4种针对Id1基因不同干扰靶点的Id1-si RNA慢病毒干扰载体(p CGSIL-GFPId1-1、p CGSIL-GFP-Id1-2、p CGSIL-GFP-Id1-3及p CGSIL-GFP-Id1-4),将其转染至293T细胞,以筛选出针对Id1基因的最有效的RNA干扰(RNAi)靶序列。再以沉默效果最优的干扰载体进一步构建慢病毒(Id1-RNAi-LV),感染人Hep G2细胞后,采用实时定量PCR法和Western blot法分别检测其Id1 m RNA及其蛋白的表达水平。结果与p CGSIL-GFP-Id1-1组、p CGSIL-GFP-Id1-2组及p CGSIL-GFP-Id1-3组比较,p CGSIL-GFP-Id1-4组的Id1蛋白表达水平最低(P<0.05),沉默效果最优。以p CGSIL-GFP-Id1-4干扰载体组建慢病毒后,测得慢病毒的病毒滴度为2.0×109 TU/m L。与空白对照组和阴性对照组比较,以组建的慢病毒感染人Hep G2细胞后,人Hep G2细胞中Id1m RNA及其蛋白的表达水平均降低(P<0.05)。结论本实验构建的特异性慢病毒可稳定地介导Id1基因的沉默。

Objective To construct the human small interfering RNA （siRNA） lentiviral vector who targeting inhibitor ofdifferentiation-1 （Id1） gene, and to detect its efficiency ofgene silence for the HepG2 cells. Methods The most effective RNA interference sequences was screened from 4 kinds of siRNA vectors targeting Idl gene （included pCGSIL-GFP-Id1-1, pCGSIL-GFP-Id1-2, pCGSIL-GFP-Id1-3, and pCGSIL-GFP-Id1-4）, who was transfected to 293T cells. The selected siRNA vector was used to build lentiviral vector （Idl-RNAi-LV） and then infected human HepG2 cells, Then the expression levels of Id1 mRNA and its protein were detected by the real time PCR and Western blot method respectively. Results Expression level of Idl protein in pCGSIL-GFP-Id1-4 group was lower than those of pCGSIL-GFP-Id1-1 group, pCGSIL-GFP-Id1-2 group, and pCGSIL-GFP-Id1-3 group （P 〈 0.05）, who had the best efficiency of gene silence. The Id1-siRNA lentiviral vector （Id1-RNAi-LV） was successfully constructed by using pCGSIL-GFP-Id1-4. The titer of lentiviral was 2.0 × 10^9 TU/mL. Results of real time-PCR and Western blot showed that, the expression levels of Idl mRNA and its protein in HepG2 cells of Id1-RNAi-LV group were lower than those of blank control group and negative control group （P〈0.05）. Conclusions The specific lentiviral can constantly downregulate the expression ofld1 gene.