miR-M11基因编辑对马立克病病毒体外复制的影响

Effect of miR‐M11 Gene Editing on Replication of Marek’s Disease Virus in Vitro

为探究病毒编码的微小RNA前体基因miR-M11对马立克病病毒(MDV)体外复制能力的影响,以MDV国内分离株HN_(3)0_(2)为研究对象,利用CRISPR/Cas9系统靶向编辑并敲除位于Mid基因簇的miRM11,构建1株miR-M11基因编辑缺失毒株HN_(3)0_(2)ΔM11(克隆号C58-8-4)。经过PCR鉴定、测序分析、间接免疫荧光试验(IFA)、实时荧光定量PCR(qPCR)以及传代稳定性分析,证实HN302编码的miRM11被精确编辑并从病毒基因组中完全缺失,且未发生回复突变。病毒增殖曲线绘制结果显示,miRM11缺失毒株HN_(3)0_(2)ΔM11的增殖速度显著高于亲本毒株HN_(3)0_(2)(P<0.05)。综上,miR-M11是MDV复制的非必需基因,且其缺失后可增强MDV的体外复制能力。

In order to explore the effect of virus‐encoded miRNA precursor miR‐M11 on the in vitro replication ability of MDV,the domestic isolate HN_(3)0_(2)of MDV was used as the parental virus,and the CRISPR/Cas9 system was used to targetedly edit and knock out miR‐M11 located in the Mid gene cluster,to construct a miR‐M11 gene editing deletion strain HN302ΔM11(clone number C58‐8‐4).After PCR identification,DNA sequencing,indirect immunofluorescence assay(IFA),real‐time quantitative PCR(qPCR),and passage stability analysis,it was confirmed that the miR‐M11 encoded by HN302 wasprecisely edited and completely deleted from the viral genome,and no reverse mutation occurred.The results of the virus proliferation curve assay showed that the proliferation rate of the miR‐M11‐deleted virus strain HN_(3)0_(2)ΔM11 was significantly higher than that of the parental virus strain HN_(3)0_(2)(P<0.05).In conclusion,miR‐M11 is a non‐essential gene for MDV replication,and its deletion can enhance the in vitro replication ability of MDV.