应用CRISPR/Cas9技术敲除Mda5基因对新城疫及传染性法氏囊病毒复制的影响

Effect of Knocking Out the Mda5 Gene by CRISPR/Cas9 Technology on the Replication of Newcastle Disease and Infectious Bursal Virus

采用CRISPR/Cas9技术敲除DF-1的黑色素瘤分化相关基因5(melanoma-differentiation-associated gene 5,Mda5),探索Mda5基因对新城疫病毒(Newcastle disease virus,NDV)和传染性法氏囊病毒(infectious bursal disease virus,IBDV)复制的影响。通过CRISPR/Cas9技术构建Mda5敲除的DF-1细胞系;实时荧光定量PCR和免疫荧光法检测病毒感染Mda5敲除细胞后病毒RNA水平、细胞病变情况及抗病毒相关基因mRNA水平等。获得Mda5基因敲除的DF-1细胞;与对照组细胞相比,细胞形态、贴壁能力和增殖能力均无显著性差异;与对照组细胞相比,Mda5基因敲除的DF-1细胞感染IBDV后,IFN-β和PKR表达显著性下调,CH25H的表达和病毒复制水平均无显著性差异;感染NDV后,IFN-β表达和病毒复制水平显著性下调,CH25H表达量显著性上调,PKR表达无显著性差异。CRISPR/Cas9技术建立Mda5敲除的DF-1中,IFN-β虽然显著应答IBDV和NDV感染,但不能显著抑制病毒的复制,说明Mda5并非抗病毒复制的关键模式识别受体。

The melanoma-differentiation-associated gene 5(Mda5)of DF-1 was knocked down using CRISPR/Cas9 technology to explore the effect of the Mda5 gene on the replication of Newcastle disease virus(NDV)and infectious bursal disease virus(IBDV)replication.CRISPR/Cas9 gene editing technology was applied to construct Mda5 knockout DF-1 cell lines,and real-time fluorescence quantitative PCR and immunofluorescence were used to detect the RNA levels of viruses,cytopathic conditions and mRNA levels of antiviral-related genes after IBDV and NDV infection of Mda5 knockout cells.Mda5 knockout DF-1 cells were obtained;there were no significant differences in cell morphology,apposition ability and proliferation ability compared to control cells.Compared to control cells,Mda5 knockout DF-1 cells infected with IBDV showed significant down-regulation of IFN-β and PKR expression,no significant differences in CH25H expression and viral replication levels.After infection with NDV,IFN-β expression and viral replication levels were significantly down-regulated,CH25H expression was significantly up-regulated,and PKR expression was not significantly different after infection with NDV.In DF-1 while applying CRISPR/Cas9 technology to knockout Mda5,though IFN-β significantly responded to IBDV and NDV infection,failed to significantly inhibit viral replication,suggesting that Mda5 is not a key pattern recognition receptor for antiviral replication.