鸭瘟病毒疫苗株UL35缺失株的构建及其免疫保护效果评价

doi:10.11843/j.issn.0366-6964.2025.08.031

摘要/Abstract

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 TCID₅₀·0.1 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，1×10⁶ TCID₅₀的rDEV-ΔVP26对30日龄麻鸭无致病作用，且1×10⁵ TCID₅₀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。

关键词: 鸭瘟病毒, VP26, 非必需基因, 鉴别诊断

Abstract:

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 TCID₅₀·0.1 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 10⁶ TCID₅₀ rDEV-ΔVP26 is safe to 30-day-old ducks, and pre-immune ducks with 10⁵ TCID₅₀ 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.

Key words: duck enteritis virus, VP26, nonessential gene, differential diagnosis

中图分类号:

S852.659.1

陈柳, 相生瑞, 云涛, 倪征, 华炯钢, 朱寅初, 张存, 叶伟成. 鸭瘟病毒疫苗株UL35缺失株的构建及其免疫保护效果评价[J]. 畜牧兽医学报, 2025, 56(8): 3933-3941.

CHEN Liu, XIANG Shengrui, YUN Tao, NI Zheng, HUA Jionggang, ZHU Yinchu, ZHANG Cun, YE Weicheng. Construction and Evaluation of Immune Protection Capacity of a Recombinant Duck Enteritis Virus Deleting UL35 Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3933-3941.

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引物名称Primers	序列Sequences
DEV-ΔVP26-for	5′- agagaacgctcttcatacaacaccacgtctacggcataacaacaaataaatgagtatgattcaata AGGATGACGACGATAAGTAGGG-3′
DEV-ΔVP26-rev	5′-aataaaagcttttatgaatctttattgaatcatactcatttatttgttgttatgccgtagacgtggCAA CCAATTAACCAATTCTGATTAG-3′
ΔVP26-JD-F	5′-gtgtgttttgtacgtatgccttgtg-3′
ΔVP26-JD-R	5′-gacgttcgcacatcaataatagct-3′

组别 Group	接种毒株 Virus	接种剂量/体积 Inoculation dose/volume	发病率/% Morbidity	死亡率/% Mortality
1缺失株Missing strain	rDEV-ΔVP26	10⁶ TCID₅₀/ 1 mL	0	0
2亲本毒株Parent strain	rDEV-EF1	10⁶ TCID₅₀/ 1 mL	0	0
3缺失株Missing strain	rDEV-ΔVP26	10⁵ TCID₅₀/ 1 mL	0	0
4亲本毒株Parent strain	rDEV-EF1	10⁵ TCID₅₀/ 1 mL	0	0
5对照Control	DMEM	1 mL	0	0

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