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

doi:10.15933/j.cnki.1004-3268.2023.01.014

Journal of Henan Agricultural Sciences ›› 2023, Vol. 52 ›› Issue (1): 134-143.DOI: 10.15933/j.cnki.1004-3268.2023.01.014

• Animal Science Veterinary Medicine • Previous Articles Next Articles

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

WANG Weidong^1,2,3，TENG Man^1,3，ZHENG Luping^1,3，LIU Jinling^1,3，ZHANG Wenkai^1,3,4，LI Linyan^1,3,4，ZHANG Zhihui^1,^2,3，FAN Jianming²，LUO Jun^1,3,4

（1.Key Laboratory of Animal Immunology，Henan Academy of Agricultural Sciences/ Ministry of Agriculture and Rural Affairs & Henan Provincial Key Laboratory of Animal Immunology，Zhengzhou 450002，China；2.College of Public Health，Zhengzhou University，Zhengzhou 450001，China；3.UK‑China Centre of Excellence for Research on Avian Diseases，Henan Academy of Agricultural Sciences，Zhengzhou 450002，China；4.College of Animal Science and Technology，Henan University of Science and Technology，Luoyang 471003，China）

Received:2022-06-01 Published:2023-01-15 Online:2023-03-09

miR-M11 基因编辑对马立克病病毒体外复制的影响

王伟东^1,2,3，滕蔓^1,3，郑鹿平^1,3，刘金玲^1,3，张文凯^1,3,4，李林燕^1,3,4,张志会^1,2,3，樊剑鸣²，罗俊^1,3,4

（1.河南省农业科学院动物免疫学重点实验室/农业农村部动物免疫学重点实验室/河南省动物免疫学重点实验室，河南郑州 450002；2.郑州大学公共卫生学院，河南郑州 450001；3.河南省农业科学院中英禽病国际研究中心，河南郑州 450002；4.河南科技大学动物科技学院，河南洛阳 471003）

通讯作者: 罗俊（1978-），男，河南罗山人，研究员，主要从事动物病毒学研究。E-mail：luojun593@aliyun.com 樊剑鸣（1971-），男，河南罗山人，副教授，主要从事分子毒理学研究。E-mail：fan5746067@126.com
作者简介:王伟东（1995-），男，河南周口人，在读硕士研究生，研究方向：分子毒理学。E-mail：wdwang136@163.com
基金资助:
国家自然科学基金项目（U21A20260）；河南省自然科学基金项目（212300410359）；河南省农业科学院自主创新项目（2022ZC65）

Abstract

Abstract: In order to explore the effect of virus‑encoded miRNA precursor miR‑M11 on the in vitro replication ability of MDV，the domestic isolate HN302 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 was precisely 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 HN302ΔM11 was significantly higher than that of the parental virus strain HN302（P<0. 05）.In conclusion，miR‑M11 is a non‑essential gene for MDV replication，and its deletion can enhance the invitro replication ability of MDV.

Key words: Marek’s disease, miR?M11, miRNA, CRISPR/Cas9, Gene editing, qPCR, Replication

摘要： 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HN302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HN302ΔM11（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HN302ΔM11的增殖速度显著高于亲本毒株HN302（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。

关键词: 马立克病, miR-M11, 微小RNA, CRISPR/Cas9, 基因编辑, qPCR, 复制

CLC Number:

S855.3

WANG Weidong, TENG Man, ZHENG Luping, LIU Jinling, ZHANG Wenkai, LI Linyan, ZHANG Zhihui, FAN Jianming, LUO Jun. Effect of miR‑M11 Gene Editing on Replication of Marek’s Disease Virus in Vitro[J]. Journal of Henan Agricultural Sciences, 2023, 52(1): 134-143.

王伟东, 滕蔓, 郑鹿平, 刘金玲, 张文凯, 李林燕, 张志会, 樊剑鸣, 罗俊. miR-M11 基因编辑对马立克病病毒体外复制的影响[J]. 河南农业科学, 2023, 52(1): 134-143.

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

miR-M11 基因编辑对马立克病病毒体外复制的影响

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