PERK干扰慢病毒载体的构建及其在人牙髓细胞中的表达

目的:构建人PERK基因的shRNA慢病毒载体,检测其对人牙髓细胞(dental pulp cells,DPCs)PERK基因表达的抑制作用.方法:针对人PERK基因的cDNA序列,设计并合成针对人PERK基因的shRNA表达序列,将其连接到载体hU6-MCS-CMV-EGFP中.测序正确后,将构建的目的载体和包装质粒共转染293T细胞,72 h后收获并浓缩得到重组慢病毒颗粒.筛选最佳感染复数(multiplicity of infection,MOI),将病毒感染人牙髓细胞,通过实时荧光定量PCR(quantitative real-time PCR,RT-PCR)和蛋白质免疫印迹(Western blot)技术检测PERK基因mRNA和蛋白的表达水平,验证干扰效果.采用SPSS24.0软件包对数据进行统计学分析.结果:成功构建LV-PERK-RNAi慢病毒载体,病毒滴度为3×108 TU/mL,最适MOI=30;RT-PCR和Western印迹法检测显示,与空白对照组相比,PERK基因的mRNA和蛋白质表达水平显著下降,在mRNA水平的表达抑制率约为63％.结论:成功构建PERK干扰慢病毒表达载体,为后续的研究创造了条件.

Construction of a lentiviral vector of RNA interference of PERK gene and identification in human dental pulp cells

PURPOSE:To construct an expression vector of a small hairpin RNA (shRNA) targeting human PERK gene and to observe gene-silencing effects of PERK in human dental pulp cells (DPCs).METHODS:According to PERK gene cDNA sequence,shRNA was designed and synthesized,which was then annealed into hU6-MCS-CMV-EGFP vector.After identified by sequencing,hU6-MCS-CMV-EGFP vector and packaging vector were co-transfected into 293 T cells.72 hours later,the recombinant lentiviruses were obtained after harvesting and concentrating.Then LV-PERK-RNAi vectors were transfected into DPCs at an appropriate multiplicity of infection.To verify the interference effect,real-time PCR and Western blot were used to detect expression levels of PERK mRNA and protein in the transfected DPCs.The data were analyzed with SPSS 24.0 software package.RESULTS:LV-PERK-RNAi vectors were successfully constructed with a high titer of 3×108 TU/mL.The results of RT-PCR and Western blot demonstrated that after infection with LV-PERK-RNAi vector at a multiplicity of infection of 30,the expression level of PERK gene in DPCs was significantly down-regulated compared with control group.At mRNA level,the interference rate was about 63％.CONCLUSIONS:An effective lentiviral shRNA expression vector targeting the PERK gene is successfully constructed and can be used for further study on the function of PERK gene.