人鼠共打靶ERβ特异性基因沉默载体的构建和鉴定

Construction and Ldentification of Silencing Vectors of Human and Mice Co-targeting ERβ-specific Gene

目的:构建人和鼠共打靶特异性ERβ基因沉默慢病毒载体。方法:检索人和小鼠ERβ基因转录本及其序列,应用SiRNA Target Finder软件设计人和小鼠共打靶ERβ基因沉默siRNA和阴性对照siRNA,合成发夹状双链shRNA,与线性化质粒PLL3.7连接构建为重组质粒(ERβ-shRNA-PLL3.7和阴性对照质粒ER-N-shRNA-PLL3.7),经鉴定后,转染人成骨肉瘤细胞MG63和小鼠成骨细胞株MC3T3-E1,经Realtime-PCR和western blot法检测重组质粒对目的基因沉默效果。应用SPSS16.0软件统计分析,检验水准为α=0.05。结果:经测序证明重组质粒中含有目的shRNA序列。分别转染小鼠成骨细胞株MC3T3-E1和人成骨肉瘤细胞MG63细胞后,无论Realtime-PCR检测ERβ基因表达,还是western blot法检测蛋白表达,均较空白对照组或阴性对照组显著降低,差异有显著性,P<0.05。结论:成功构建了人和小鼠共同高效打靶的ERβ基因沉默慢病毒载体质粒,并包装成假病毒颗粒,为利用小鼠作为动物模型研究人基因功能或开发基因药物奠定了基础。

Objective：To construct the silencing lentiviral vectors human and mice co-targeting ERβ-specific gene of human and mice.Methods：Retrieving the transcripts and sequence of the ERβgene of human and mice.Using the software SiRNA Target Finder to design human and mice co-targeting ERβ gene silencing siRNA and negative control,and compounding double stranded shRNA in shape of stem loop,being connected with linearized plasmid PLL3.7 by T4 DNA ligase to reconstruct as a recombinant plasmid （ERβ-shRNA-PLL3.7 and negative control plasmid ER-N-shRNA-PLL3.7）, which were identified and transfected into human osteosarcoma cells MG63 and mouse osteoblastic cell line MC3T3-E1 cells. The testing ways of Realtime-PCR and western blot were used to test gene silencing effect of the recombinant plasmid.The SPSS1 6.0 software was used for statistical analysis,and the testing level was set at P 〈0.05.Results：It was confirmed by sequencing that the recombinant plasmid contained the shRNA sequence.After transfecting into mice osteoblastic cell line of MC3T3-E1 and human osteosarcoma cells of MG63,whether the ERβgene expression detected by Realtime-PCR,or Protein expression detected by the western blot,were decreased significantly compared with the control group or the negative control group,and there was a prominent difference,P 〈0.05.Conclusion：successfully constructed the ERβ-specific gene silencing lentiviral vectors of human and mice co-targeting effectively,and they were packaged into pseudoviral particles,which laid a foundation for the study of man gene function by using mouse as animal model or the development of gene-based drugs.