PRPS2基因shRNA质粒的构建与表达

Construction and its expression of PRPS2 shRNA plasmid

目的构建磷酸核糖焦磷酸合成酶2(phosphoribosyl pyrophosphate synthetase 2,PRPS2)基因shRNA质粒,为探讨PRPS2基因功能奠定基础。方法设计并合成PRPS2基因shRNA,将其分别与真核表达载体GV102重组为shRNA质粒,分别命名为GV102-PRPS2-1、GV102-PRPS2-2、GV102-PRPS2-3,分别转染这三种载体的细胞设为实验组1、2、3;阴性对照载体命名为GV102-PRPS2-0,转染该载体设为阴性对照组;分别测序鉴定。常规转染人结肠癌HCT116细胞后采用RT-PCR、Western blot检测细胞PRPS2基因的表达水平,筛选干扰效果最佳shRNA载体。结果设计并合成的PRPS2干扰载体,经测序鉴定序列正确。转染HCT116细胞后在72 h内发绿色荧光的细胞数量随时间的增加而增强,转染效率均介于40%-50%。RT-PCR和Western blot结果显示,转染shRNA质粒的3个实验组PRPS2表达均较阴性对照组显著下降(P<0.05),其中GV102-PRPS2-3使细胞中PRPS2的mRNA(0.27±0.05)水平下降73%、蛋白(0.30±0.04)水平下降70%,干扰效果优于GV102-PRPS2-1(mRNA:0.61±0.03,蛋白:0.37±0.06)和GV102-PRPS2-2(mRNA:0.89±0.02,蛋白:0.84±0.05)。结论本研究利用RNAi技术下调了人结肠癌HCT116细胞中PRPS2基因的表达,为深入探讨PRPS2表达下调后对细胞行为的影响奠定了研究基础。

Objective To construct a series of plasmids containing the shRNA for phosphoribosyl pyrophosphate synthetase 2 and provide a basis for the study of the function of PRPS2 gene.Methods A series of shRNA for PRPS2 were designed and synthesized,and then recombined with eukaryotic expression vector GV102 as shRNA recombinant plasmids（experiment groups） and identified by sequencing.The recombinant plasmids were named as GV102-PRPS2-1,GV102-PRPS2-2,GV102-PRPS2-3,respectively.The plasmids were transfected to HCT116 cells.RT-PCR and Western blot were performed to detect the expression level of PRPS2 for selecting the best plasmid.Results The sequences of shRNA recombinant plasmids were confirmed correct.The HCT116 cells expressing green fluoresce increased time-dependently in 72 h after transfection and the transfection rate was 40％-50％.The RT-PCR and Western blot results showed that the relative expression quantities of PRPS2 in experiment groups were decreased significantly compared with negative control group （P ＜ 0.05）.The mRNA expression of PRPS2 in GV102-PRPS2-3 group decreased by 73％ to 0.27 ± 0.05 and PRPS2 protein level decreased by 70％ to 0.30 ± 0.04,and its interference effect was better than GV102-PRPS2-1 （mRNA：0.61 ±0.03,protein：0.37 ±0.06） and GV102-PRPS2-2（mRNA：0.89 ±0.02,protein：0.84 ±0.05）.Conclusion The study makes it successful to down-regulate the expression of PRPS2 in HCT116 cells by RNAi and lay a foundation to study the effect of down-regulation of PRPS2 on cells.