畜牧兽医学报2024,Vol.55Issue(12) :5672-5683.DOI:10.11843/j.issn.0366-6964.2024.12.030

一株马立克病病毒特超强变异株meq基因编辑缺失候选疫苗毒株的构建与鉴定

Development and Pathogenicity Analysis of a meq-gene-edited Candidate Marek's Disease Vaccine Strain Generated from a Hypervirulent MDV Variant

张多 滕蔓 张卓 刘金玲 郑鹿平 各思雨 韩放 罗琴 柴书军 赵东 余祖华 罗俊
畜牧兽医学报2024,Vol.55Issue(12) :5672-5683.DOI:10.11843/j.issn.0366-6964.2024.12.030

一株马立克病病毒特超强变异株meq基因编辑缺失候选疫苗毒株的构建与鉴定

Development and Pathogenicity Analysis of a meq-gene-edited Candidate Marek's Disease Vaccine Strain Generated from a Hypervirulent MDV Variant

张多 1滕蔓 2张卓 3刘金玲 2郑鹿平 2各思雨 3韩放 1罗琴 4柴书军 2赵东 2余祖华 5罗俊3
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作者信息

  • 1. 河南科技大学动物科技学院,功能微生物与畜禽健康实验室,洛阳 471003;河南省农业科学院动物疫病防控研究所,中英禽病国际研究中心,郑州 450002;龙湖现代免疫实验室,郑州 450046
  • 2. 河南省农业科学院动物疫病防控研究所,中英禽病国际研究中心,郑州 450002;龙湖现代免疫实验室,郑州 450046
  • 3. 河南省农业科学院动物疫病防控研究所,中英禽病国际研究中心,郑州 450002;龙湖现代免疫实验室,郑州 450046;郑州大学公共卫生学院,郑州 450001
  • 4. 河南牧业经济学院动物医药学院,郑州 450046
  • 5. 河南科技大学动物科技学院,功能微生物与畜禽健康实验室,洛阳 471003
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摘要

马立克病(MD)是由马立克病病毒(MDV)感染引起的一种重要的家禽免疫抑制病与肿瘤病,该病可用疫苗进行预防和控制,但在长期免疫压力下MDV的毒力持续增强,经典MD疫苗已难以对当前流行的特超强MDV(HV-MDV)变异株提供良好的免疫保护,亟需研发新一代MD高效疫苗.本文以HV-MDV变异株HNSQ01为亲本毒株,在CEF上连续传代之后,利用CRISPR/Cas9系统对其meq基因进行编辑,通过一系列试验鉴定和分析,最终建立一株meq基因编辑缺失的MD疫苗候选毒株,命名为SQ01Δmeq.1日龄SPF鸡攻毒试验结果显示,在77 d试验周期内,亲本毒株HNSQ01不仅严重抑制感染鸡生长并导致免疫抑制,而且导致100%的MD发病率、100%致死率及80%的肉眼观察肿瘤发生率;但SQ01Δmeq未引起感染鸡的生长及免疫抑制,而且MD发病率、致死率及肿瘤发生率均为0%,表明其对宿主无致病性,生物安全性良好.本研究建立的meq基因编辑缺失MDV毒株SQ01Δmeq,为后续研发新型高效的MD疫苗奠定了重要基础.

Abstract

Marek's disease(MD),caused by Marek's disease virus(MDV),is one of the most im-portant immunosuppressive and neoplastic diseases.It can be prevented and controlled with MD vaccines,but the virulence of epidemic MDV strains persistently increases under the highly im-mune pressure of widely used MD vaccines.Recent studies have found that the classical MD vac-cines are no longer able to provide ideal immune protection for the current prevalent hypervirulent MDV(HV-MDV)variant,which implies the urgent need to develop a new generation of highly effective MD vaccines.Presently,we have used the newly isolated HV-MDV variant HNSQ01 as the parental virus,first passaged on CEF monolayers in vitro,to generate a meq gene-edited and knocked out mutant of SQ01Δmeq utilizing the CRISPR/Cas9-based gene editing technology.A series of experiments and analysis have demonstrated a stable MD vaccine candidate strain of SQ01Δmeq has been successfully generated.Furthermore,the pathogenicity analysis was per-formed with 1-day-old specific-pathogen-free(SPF)chickens for virus challenge experiments.During the 77-days animal experiments,the parental HNSQ01 had caused serious atrophy of host immune organs and inhibited the growth of virus-challenged birds,together with the 100%MD morbidity,100%mortality and 80%gross tumor occurrence.However,SQ01Δmeq had not caused the immunosuppression or inhibited the growth of virus-challenged birds,together with the 0%rates of MD morbidity,mortality and tumor occurrence.Our data indicate that the gener-ation of SQ01Δmeq provides an important basis for the future research and development of novel efficient genetically engineered MD vaccines.

关键词

马立克病/MDV/meq/CRISPR/Cas9/基因编辑/基因缺失/新型疫苗

Key words

Marek's disease/MDV/meq gene/CRISPR/Cas9/Gene editing/gene deletion/novel vaccine

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出版年

2024
畜牧兽医学报
中国畜牧兽医学会

畜牧兽医学报

CSTPCD北大核心
影响因子:0.729
ISSN:0366-6964
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