利用CRISPR/Cas9技术敲除TLR3基因及其影响NDV复制的研究

Knockout of TLR3 gene and its function study by CRISPR/Cas9 technology

TLR3是Toll样受体家族成员之一,在天然免疫与适应性免疫应答中均发挥重要作用。为探究TLR3在NDV感染中的作用机制,本研究针对犬TLR3基因设计了2个sgRNA,构建重组质粒,转染MDCK细胞,经PCR、测序、Western-blot检测敲除情况,CCK-8方法检测细胞增殖速度;新城疫病毒La Sota毒株感染细胞,荧光定量PCR分析病毒增殖水平和TLR3、TLR7、MDA5、IFN-β和IFIT1的m RNA水平。结果,筛选出1株TLR3基因缺失7 bp的MDCK细胞。Western-blot结果显示,TLR3蛋白不表达,其增殖速度与野生型MDCK细胞无显著差异。新城疫病毒La Sota毒株感染MDCK-WT细胞后,TLR3的m RNA含量在四个时间点均显著上调(P<0.01),表明新城疫病毒La Sota毒株可激活TLR3。再以新城疫病毒La Sota毒株感染MDCK-TLR3-/-细胞,IFN-β和IFIT1的m RNA含量在感染后第12小时和第24小时显著降低(P<0.01),病毒拷贝数在感染后第6、12和24小时显著高于WT组(P<0.01),表明TLR3在NDV介导的IFN-β反应和IFIT1的表达中具有重要作用,能够抑制NDV的复制。TLR7和MDA5的m RNA含量在感染后第6小时和第12小时显著上调(P<0.01),IFN-β和IFIT1的m RNA含量在感染后第48小时表达显著上调(P<0.01),NDV的拷贝数在第48小时在两组间显著性差异消失,提示TLR3缺失后TLR7和MDA5对其具有代偿作用。上述研究结果对NDV介导的天然免疫应答及疾病防控等研究具有重要意义。

TLR3 is a member of the Toll-like receptor family and plays an important role in both innate and adaptive immune responses.In order to explore the mechanism of TLR3 in Newcastle disease virus(NDV)infection,two sgRNAs were designed for canine TLR3 gene,and the recombinant plasmid was constructed and transfected into MDCK cells.Detected by PCR,sequenced and Western-blot,and the cell proliferation rate was detected by CCK-8 method.The Newcastle disease virus La Sota strain infected,and the virus proliferation level and m RNA level of TLR3,TLR7,MDA5,IFN-βand IFIT1 were analyzed by real-time PCR.In result,one strain of MDCK cells with 7 bp deletion of TLR3 gene was screened.Western-blot showed that TLR3 protein was not expressed,and the proliferation rate was not significantly different from MDCK-WT cells.After MDCK-WT cells infected by Newcastle disease virus La Sota strain,the m RNA level of TLR3 was significantly up-regulated at four time points(P<0.01),indicating that Newcastle disease virus La Sota strain can activate TLR3.The m RNA level of IFN-βand IFIT1 were significantly decreased at 12 h and 24 h post-infectin(P<0.01)in the MDCK-TLR3-/-cells infected with Newcastle disease virus La Sota strain,the virus copy number was significantly higher at6,12 and 24 h post-infectin.This indicates TLR3 plays an important role in NDV-mediated IFN-βresponse and expression of IFIT1,and can inhibit NDV replication.The m RNA level of TLR7 and MDA5 were significantly up-regulated at 6 h and 12 h post-infectin(P<0.01),and IFN-βand IFIT1 were significantly up-regulated at 48 h post-infectin(P<0.01).The significant difference of virus copy number disappeared at 48 h post-infectin,suggesting TLR7 and MDA5 have Compensation on TLR3 deletion.The above-mentioned results are of great significance for the study of NDV-mediated natural immune response and disease prevention and control.