人β-COP-shRNA 慢病毒载体构建及干扰效应检测

摘要

[Abstract ] Objective The purpose of this study was to construct a short hairpin RNA (shRNA) interference lentiviral vector targeting the humanβ-COP gene and to evaluate its inhibitory effect on β-COP in THP-1 cells. Methods We designed and synthesized 4 humanβ-COP-specific oligonucleotide sequences and inserted them into the pGMLV-SC1 vector to construct a recombinant vector fol-lowed by transfection of HEK 293T cells with the recombinant vector and Lenti-HG Mix to produce lentiviruses and detect the viral con-tent.After infecting the THP-1 cells with the packaged lentiviruses , we analyzed the inhibitory effect of β-COP-shRNA on the β-COP gene by quantitative PCR and Western blot . Results Sequencing confirmed that the β-COP-specific oligonucleotide sequences were in-serted into the lentiviral vector and the lentiviruses were packaged in the transfected HEK 293T cells, with the final viral content of 1 × 109 TU/mL.Quantitative PCR showed that the 4 β-COP-shRNA vectors significantly decreased the mRNA expression of β-COP (P<0.01), with interference rates of 16.9 %,32.5%, 74.0%, and 50.3%, respectively.Western blot also indicated their inhibitory effect on the protein expression of β-COP, with an inhibition rate of 76.4% onβ-COP-shRNA3. Conclusion Lentiviral shRNA interference vectors targeting human β-COP were constructed successfully , which could suppress the expression of the human β-COP gene.%目的：采用常规转染试剂难以将人β-COP-shRNA转染至THP-1细胞。构建人β-COP特异性的shRNA慢病毒载体，并测定其对靶基因β-COP的抑制效应。方法设计合成针对人β-COP基因靶点特异的4对shRNA寡聚单链DNA，插入pGMLV-SC1载体中，构建慢病毒重组载体。将重组载体和包装质粒共转染HEK 293 T细胞，包装产生慢病毒并测定滴度。用慢病毒感染THP-1细胞，定量PCR 和Western blot 检测干扰载体对β-COP基因的干扰效果。结果测序证实，β-COP基因的shRNA寡聚核苷酸序列已插入慢病毒载体，转染HEK 293T细胞，经包装得到4种慢病毒，病毒滴度为1×109 TU／mL。定量PCR分析显示，4种β-COP-shRNA 慢病毒感染的 THP-1细胞中β-COP基因 mRNA 表达量分别为0．831±0．065、0．675±0．079、0．260±0．050、0．497±0．067，较对照（1．000±0．000）明显升高（ P＜0．01）；其抑制率分别为16．9％、32．5％、74．0％和50．3％。 Western blot结果表明，4种干扰载体均可抑制β-COP蛋白表达，其中β-COP-shRNA 3的沉默效率为76．4％。结论成功构建了人β-COP-shRNA干扰载体，证实了干扰载体对靶基因有较好的沉默效果。