摘要
为研究鸭瘟病毒(duck enteritis virus,DEV)UL35基因缺失DEV疫苗株的生物学特性和对强毒株攻击的免疫保护效果,本研究在鸭瘟病毒疫苗株感染性克隆pDEV-EF1基础上,通过“Red E/T两步重组”技术,构建了UL35基因缺失的pDEV-ΔVP26突变体克隆,并转染鸡胚成纤维细胞(CEFs)获得了重组病毒rDEV-ΔVP26,进而对重组病毒细胞生物学特性和免疫保护能力进行了评估。病毒一步生长曲线测定表明,rDEV-ΔVP26的滴度从24~84 h稳步上升,达到峰值10^(5.36)TCID_(50)·0.1 mL^(-1)。在此期间,rDEV-ΔVP26滴度较亲本毒株rDEV-EF1相比有所降低。蚀斑面积测定发现rDEV-ΔVP26蚀斑面积较亲本株rDEV-EF1减少了10.60%,说明UL35基因缺失会轻微影响病毒扩散并降低病毒滴度。动物试验表明,1×10^(6)TCID_(50)的rDEV-ΔVP26对30日龄麻鸭无致病作用,且1×10^(5)TCID_(50)滴度的rDEV-ΔVP26免疫组在强毒株攻击下的保护率与相同剂量的rDEV-EF1对照组一致。该研究表明,UL35为DEV复制非必需基因,且当接种合适剂量的病毒液时,UL35缺失不影响鸭瘟病毒疫苗株的免疫保护效果。该研究为研发用于区分疫苗免疫和野毒感染的DEV鉴别诊断疫苗奠定了基础。
This study aimed to study the biological characteristics and the immunoprotective effect of DEV vaccine strain with UL35 gene deletion.A UL35 gene-deletion virus strain was constructed based on the bacterial artificial chromosome(BAC)clone pDEV-EF1 which carries DEV vaccine strain full-length genome.The BAC clone pDEV-ΔVP26 of UL35 gene deletion was generated by two-step Red/ET recombination in E.coli.The recombinant virus rDEV-ΔVP26 was rescued from chicken embryo fibroblasts(CEFs)by calcium phosphate precipitation.And then the biological characteristics and immunoprotective capacity of recombinant virus were evaluated.Growth curves show that the virus titer of rDEV-ΔVP26 was stable increasing from 24 to 84 h,up to the peak value 10^(5.36)TCID_(50)·0.1 mL^(-1).During this period,the titer of rDEV-ΔVP26 was slightly reduced compared to the parental strain rDEV-EF1,and the plaque size of rDEV-ΔVP26 was decreased 10.60%compared to the parental virus.Animal experiments showed that 10^(6)TCID_(50)rDEV-ΔVP26 is safe to 30-day-old ducks,and pre-immune ducks with 10^(5)TCID_(50)rDEV-ΔVP26 could completely protect ducks from high virulent DEV challenge,this is similar to the rDEV-EF1 control group.The results showed that UL35 was non-essential gene of DEV,and absence of UL35 did not affect the immune protection capacity of DEV vaccine strain.These studies have laid a foundation for developing differential diagnosis vaccine between infected and vaccinated animals.
作者
陈柳
相生瑞
云涛
倪征
华炯钢
朱寅初
张存
叶伟成
CHEN Liu;XIANG Shengrui;YUN Tao;NI Zheng;HUA Jionggang;ZHU Yinchu;ZHANG Cun;YE Weicheng(State Key Laboratory for Quality and Safety of Agro-Products,Institute of Animal Husbandry and Veterinary Sciences,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;Nanjing Vazyme Biotech Co.,Ltd.,Nanjing 210046,China)
出处
《畜牧兽医学报》
北大核心
2025年第8期3933-3941,共9页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金面上项目(32473008)。
关键词
鸭瘟病毒
VP26
非必需基因
鉴别诊断
duck enteritis virus
VP26
nonessential gene
differential diagnosis
