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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3×10~8 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。
OBJECTIVE: To construct a shRNA lentivirus vector containing human PERK gene and investigate its inhibitory effect on PERK gene expression in human dental pulp cells (DPCs). Methods: According to the cDNA sequence of human PERK gene, the shRNA expression sequence targeting human PERK gene was designed and synthesized and ligated into vector hU6-MCS-CMV-EGFP. After sequencing correctly, 293T cells were co-transfected with the constructed vector and packaging plasmid, and then harvested and concentrated after 72 h to obtain recombinant lentivirus particles. The optimal multiplicity of infection (MOI) was screened and infected with human dental pulp cells. The PERK gene mRNA was detected by quantitative real-time PCR (RT-PCR) and Western blot And protein expression levels, to verify the interference effect. SPSS24.0 software package for statistical analysis of the data. Results: The LV-PERK-RNAi lentiviral vector was successfully constructed, the virus titer was 3 × 10-8 TU / mL and the optimal MOI was 30. RT-PCR and Western blotting showed that compared with the blank control group, PERK Gene mRNA and protein expression levels decreased significantly, the expression level at the mRNA level of inhibition was about 63%. Conclusion: The successful construction of PERK lentiviral expression vector, which created the conditions for the follow-up study.