小鼠Pim-3基因siRNA慢病毒载体的构建及表达

Construction of Lentivirus Expressing siRNA Targeted Mouse Pim-3 Gene

目的:本研究拟构建针对Pim-3基因的siRNA慢病毒载体,诱导靶细胞中Pim-3基因沉默。方法:分析Pim-3 mRNA序列特征,根据siRNA设计原则,设计并合成特异性针对小鼠Pim-3的siRNA靶DNA序列及阴性对照序列。用限制性核酸内切酶将pGCSIL-GFP载体线性化,将寡DNA片段退火成含有粘性末端的DNA双链后,与慢病毒骨架质粒连接转化至大肠杆菌中进行重组,运用PCR技术筛选阳性克隆并测序,得到pSC-Pim-3 shRNA质粒。采用脂质体将pSC-Pim-3 shRNA质粒和辅助包装质粒共转染到HEK293细胞中包装形成慢病毒。采用倍比稀释法测定慢病毒滴度。慢病毒感染NIH3T3细胞,运用实时定量PCR技术检测Pim-3基因mRNA表达水平,运用蛋白免疫印迹技术检测Pim-3蛋白表达水平。结果:酶切后获得7.5 kb的pGCSIL-GFP片段。DNA片段成功连接到pGCSIL-GFP载体;重组慢病毒的滴度约为2×109TU/mL。慢病毒能够高效感染靶细胞,并显著抑制内源性Pim-3基因的mRNA(抑制率超过70%)和蛋白(抑制率超过80%)表达。结论:针对Pim-3基因靶序列CCTCTTCGACT-TCATCACT的shRNA重组慢病毒能够有效沉默靶细胞Pim-3基因。

Objective To construct a lentivirus expressing siRNA targeted mouse Pim-3 gene.Methods We first identified and designed the oligonucleotides to generate the siRNA.pGCSIL-GFP vector was digested with Age I /EcoR I enzymes.The recombinant pGCSIL-GFP vectors were transformed into competent cells.Well-isolated colonies were picked up and analyzed by using PCR technology.The positive colony was further amplified and purified for further experiments.The three packaging plasmids were transfected into 293T cell line to produc lentivirus stock.The titer of lentiviral vectors were calculated based on GFP expression on 293T cell.The real-time PCR method was used to determine the knock-down efficiency on Pim-3 gene expression in NIH3T3 cell line.The expression of Pim-3 protein in NIH3T3 cell was verified by western blot.Results Four siRNA sequences for Pim-3 were tested.Digestion of pGCSIL-GFP vector with Age I /EcoR I produced 7.5-kb bands.The DNA fragments were ligated into pGCSIL-GFP vector.The titer of lentiviral vectors were about 2×109 TU/mL.The mRNA expession of Pim-3 was significantly decreased(more than 70% reduction) by lentiviral vector infection.The protein expession of Pim-3 was significantly decreased(more than 80% reduction) by lentiviral vector infection.Conclusion We successfully constructed a lentivirus expressing siRNA targeted mouse Pim-3 gene.