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

doi:10.11843/j.issn.0366-6964.2024.12.030

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (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

张多¹^,²^,³(), 滕蔓²^,³, 张卓²^,³^,⁴, 刘金玲²^,³, 郑鹿平²^,³, 各思雨²^,³^,⁴, 韩放¹^,²^,³, 罗琴⁵, 柴书军²^,³, 赵东²^,³, 余祖华¹^,*(), 罗俊²^,³^,⁴^,*()

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
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
3. 龙湖现代免疫实验室，郑州 450046
4. 郑州大学公共卫生学院，郑州 450001
5. 河南牧业经济学院动物医药学院，郑州 450046

收稿日期:2024-01-21 出版日期:2024-12-23 发布日期:2024-12-27
通讯作者: 余祖华,罗俊 E-mail:16696657142@163.com;yzhd05@163.com;luojun593@aliyun.com
作者简介:张多(1998-)，女，河南永城人，硕士生，主要从事动物疫病防控技术研究，E-mail: 16696657142@163.com
基金资助:
国家自然科学基金(U21A20260)；河南省杰出青年科学基金(232300421009)；河南省农业科学院自主创新项目(2024ZC096)

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

ZHANG Duo¹^,²^,³(), TENG Man²^,³, ZHANG Zhuo²^,³^,⁴, LIU Jinling²^,³, ZHENG Luping²^,³, GE Siyu²^,³^,⁴, HAN Fang¹^,²^,³, LUO Qin⁵, CHAI Shujun²^,³, ZHAO Dong²^,³, YU Zuhua¹^,*(), LUO Jun²^,³^,⁴^,*()

1. Laboratory of Functional Microbiology and Animal Health, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, China
2. Institute for Animal Health & UK-China Center of Excellence for Research on Avian Disease, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
3. Longhu Laboratory, Zhengzhou 450046, China
4. College of Public Health, Zhengzhou University, Zhengzhou 450001, China
5. College of Veterinary Medicine, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China

Received:2024-01-21 Online:2024-12-23 Published:2024-12-27
Contact: YU Zuhua, LUO Jun E-mail:16696657142@163.com;yzhd05@163.com;luojun593@aliyun.com

摘要/Abstract

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。

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

Abstract:

Marek's disease (MD), caused by Marek's disease virus (MDV), is one of the most important 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 immune pressure of widely used MD vaccines. Recent studies have found that the classical MD vaccines 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 performed 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 generation of SQ01Δmeq provides an important basis for the future research and development of novel efficient genetically engineered MD vaccines.

Key words: Marek's disease, MDV, meq gene, CRISPR/Cas9, Gene editing, gene deletion, novel vaccine

中图分类号:

S852.659.1

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. 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[J]. 畜牧兽医学报, 2024, 55(12): 5672-5683.

ZHANG Duo, TENG Man, ZHANG Zhuo, LIU Jinling, ZHENG Luping, GE Siyu, HAN Fang, LUO Qin, CHAI Shujun, ZHAO Dong, YU Zuhua, LUO Jun. Development and Pathogenicity Analysis of a meq-gene-edited Candidate Marek's Disease Vaccine Strain Generated from a Hypervirulent MDV Variant[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5672-5683.

图/表 7

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引物名称 Primer name	序列(5’→3’) Sequence	扩增长度/bp Amplicon/bp
meq-9F	TGCTGGAATGTTAAGAATAAATTCCGCAC	1 121
meq-2R	TCAGGGTCTCCCGTCACCTGGAAACCACCA
yg-gB-F	TCTAGGGCATGGCACACGAC	125
yg-gB-R	GAATACGGAAACACAGAGCGG
yg-pp38-F	CCGAAAGACAAAACCCAAAT	129
yg-pp38-R	ATGTAACCAGCATATAAGAACGC
yg-Meq-F	CGCAGGAAGCAGACGGACTA	157
yg-Meq-R	CCATAGGGCAAACTGGCTCAT
yg-ICP4-F	GCACTGCATTCCGAGAGTCAT	161
yg-ICP4-R	TTGGGAATTTGGAGGGCG
yg-OVO-F	AAGCAAGAGAAATGGGCTGAT	133
yg-OVO-R	AGGAGGGGAAGACATCCAGTA
yg-RLORF4-F	TGCTTGTTTTGGGTAATTGGTC	275
yg-RLORF4-R	TACTGGAACACAAGACTATGAGGAC
yg-RLORF5a-F	AATACCTCATCGCAGAGACGC	162
yg-RLORF5a-R	CTCGTTCCGTTCGCTCTTTC

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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

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