生物技术通报 ›› 2022, Vol. 38 ›› Issue (11): 90-96.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0087

• 技术与方法 • 上一篇    下一篇

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

钟菁(), 孙玲玲, 张姝, 蒙园, 支怡飞, 涂黎晴, 徐天鹏, 濮黎萍, 陆阳清()   

  1. 广西大学动物科学技术学院,南宁 530004
  • 收稿日期:2022-01-19 出版日期:2022-11-26 发布日期:2022-12-01
  • 作者简介:钟菁,女,硕士研究生,研究方向:动物繁殖新技术;E-mail:1482144912@qq.com
  • 基金资助:
    国家重点研发计划项目(2021YFD1300100);广西重点研发计划项目(桂科AB21220005);巴马县人才科技计划项目(巴人科20210004)

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

ZHONG Jing(), SUN Ling-ling, ZHANG Shu, MENG Yuan, ZHI Yi-fei, TU Li-qing, XU Tian-peng, PU Li-ping, LU Yang-qing()   

  1. College of Animal Science and Technology,Guangxi University,Nanning 530004
  • Received:2022-01-19 Published:2022-11-26 Online:2022-12-01

摘要:

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-βPKR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。

关键词: Mda5, 新城疫病毒, 传染性法氏囊病毒, CRISPR/Cas9, 基因敲除

Abstract:

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.

Key words: Mda5, Newcastle disease virus, infectious bursa disease virus, CRISPR/Cas9, knockout