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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 explore the silencing effects of lentivirus with small interfering RNA (si RNA) lentiviral vector on the gene silencing of Id1 in human Hep G2 cells. Methods Four kinds of Id1-si RNA lentiviral vectors (p CGSIL-GFPId1-1, p CGSIL-GFP-Id1-2, p CGSIL-GFP-Id1-3 and p CGSIL-GFP) targeting different targets of Id1 gene were constructed -Id1-4), which was transfected into 293T cells to screen out the most effective RNA interference (RNAi) target sequence for Id1 gene. Id1-RNAi-LV was further constructed by using the silencing vector with the best silencing effect. After being infected into Hep G2 cells, the expression of Id1 m RNA and its protein was detected by real-time quantitative PCR and Western blot respectively. Results Compared with p CGSIL-GFP-Id1-1 group, p CGSIL-GFP-Id1-2 group and p CGSIL-GFP-Id1-3 group, the expression level of Id1 protein in p CGSIL-GFP-Id1-4 group was the lowest <0.05), the best effect of silence. After the lentivirus was constructed with the p CGSIL-GFP-Id1-4 interfering vector, the lentivirus titer was 2.0 × 10 9 TU / m L. Compared with the blank control group and the negative control group, the expression of Id1mRNA and its protein in human Hep G2 cells was decreased after infection with Hep G2 cells (P <0.05). Conclusion The constructed specific lentivirus can stably mediate Id1 gene silencing.