ANG-shRNA慢病毒载体干扰效率的筛选

发布时间：2018-07-06 21:08

本文选题：ANG + ANG-shRNA慢病毒载体　；参考：《基因组学与应用生物学》2017年10期

【摘要】：通过干扰血管生成素(ANG)mRNA的表达,揭示ANG在糖尿病视网膜病变过程中的作用,本研究根据NCBI中查询的ANG序列设计阴性对照序列(NC)和3条ANG-shRNA序列(ANG-shRNA-1,ANG-shRNA-2,ANG-shRNA-3)构建至慢病毒载体GV248中,并进行病毒包装,转染至293T细胞进行滴度的测定。将包装好的慢病毒感染大鼠肝癌细胞(RH-35),测定感染效率,提取RNA,通过实时荧光定量PCR(qPCR)技术筛选出干扰效率最高的ANG-shRNA序列。结果显示,通过qPCR对3条序列慢病毒干扰效率的筛选发现,NC组ANG的表达高于空白对照组(p0.01),ANG-shRNA-1、ANG-shRNA-2和ANG-shRNA-3组分别与NC组相比ANG表达均下降至99%、95%和94%。最终结果表明,在mRNA水平上3条ANG-shRNA序列干扰效率均高于90%,其中ANG-shRNA-1序列的干扰效率最高达到99%,因此3条干扰序列均可用到活体实验中。
[Abstract]:By interfering with the expression of angiopoietin (Ang) mRNA and revealing the role of Ang in diabetic retinopathy, we constructed the lentiviral vector GV248 by designing a negative control sequence (NC) and three NG-AshRNA sequences (ANG-shRNA-1, ANG-shRNA-2NG-shRNA-3) according to the ANG sequence queried in NCBI. The virus was packaged and transfected into 293T cells for titer determination. The infected rat hepatoma cells (RH-35) were infected with lentivirus, the infection efficiency was measured, the RNAs were extracted, and the ANG-shRNA sequence with the highest interference efficiency was screened by real-time fluorescent quantitative PCR (qPCR). The results showed that the expression of Ang in NC group was significantly higher than that in control group (p0.01). Compared with NC group, the expression of Ang in ANG-shRNA-1 and ANG-shRNA-3 group was significantly lower than that in NC group (95% and 94%, respectively). The results showed that the interference efficiency of three ANG-shRNA sequences was higher than that of 90 at mRNA level, and the highest interference efficiency of ANG-shRNA-1 sequence was 99, so all three interference sequences could be used in vivo.
【作者单位】：华北理工大学生命科学学院;华北理工大学基础医学院;华北理工大学附属医院;华北理工大学实验动物中心;
【基金】：河北省科技厅科技支撑计划项目(14277727D)资助
【分类号】：R587.2

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