基于F和HN蛋白的基因Ⅶ型新城疫病毒嵌合疫苗的构建及免疫效力评估

doi:10.16431/j.cnki.1671-7236.2022.11.025

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (11): 4346-4355.doi: 10.16431/j.cnki.1671-7236.2022.11.025

基于F和HN蛋白的基因Ⅶ型新城疫病毒嵌合疫苗的构建及免疫效力评估

李丽^1,2,3, 王国康^1,2,3,4, 商雨^1,2,3, 徐英英^1,2,3, 冯贺龙^1,2,3, 汪宏才^1,2,3, 张蓉蓉^1,2,3, 曾哲^1,2,3, 罗青平^1,2,3, 邵华斌^1,2,3, 曾驰⁴, 温国元^1,2,3

1. 湖北省农业科学院畜牧兽医研究所, 武汉 430064;
2. 农业农村部畜禽细菌病防治制剂创制重点实验室, 武汉 430064;
3. 畜禽病原微生物学湖北重点实验室, 武汉 430064;
4. 武汉轻工大学生物与制药工程学院, 武汉 430023

收稿日期:2022-04-01 出版日期:2022-11-05 发布日期:2022-11-04
通讯作者: 温国元 E-mail:wgy_524@163.com
作者简介:李丽,E-mail:734267021@qq.com;王国康,E-mail:965012690@qq.com。
基金资助:
湖北省自然科学基金杰出青年人才项目（2019CFA071）；国家自然科学基金面上项目（31873018）；国家现代农业产业技术体系（CARS-41-G13）；湖北省农业科技创新中心项目（2019-620-000-001-017）；湖北省科技创新项目重大专项（2020ABA016）

Construction and Immune Efficacy Evaluation of a Genotype Ⅶ Newcastle Disease Virus Chimeric Vaccine Based on F and HN Proteins

LI Li^1,2,3, WANG Guokang^1,2,3,4, SHANG Yu^1,2,3, XU Yingying^1,2,3, FENG Helong^1,2,3, WANG Hongcai^1,2,3, ZHANG Rongrong^1,2,3, ZENG Zhe^1,2,3, LUO Qingping^1,2,3, SHAO Huabin^1,2,3, ZENG Chi⁴, WEN Guoyuan^1,2,3

1. Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan 430064, China;
2. Key Laboratory of Prevention and Control Agents for Animal Bacteriosis, Ministry of Agriculture and Rural Affairs, Wuhan 430064, China;
3. Hubei Provincial Key Laboratory of Animal Pathogenic Microbiology, Wuhan 430064, China;
4. School of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China

Received:2022-04-01 Online:2022-11-05 Published:2022-11-04

摘要/Abstract

摘要： 【目的】试验旨在构建一种基因Ⅶ型新城疫病毒（Newcastle disease virus，NDV）嵌合疫苗，并对其免疫效力进行评估。【方法】利用反向遗传学技术，以含有禽偏禽腮腺炎病毒2型（Avian metaavulavirus-2，AMAV-2） Y2株基因组的重组质粒pT7-Y2为模板，将Y2株的F和HN蛋白的胞外区替换为基因Ⅶ型NDV HB0901株的F和HN蛋白的胞外区，将HB0901株的F蛋白裂解位点突变为LaSota弱毒株的F蛋白裂解位点，构建嵌合重组病毒rY2-FHNR株。对rY2-FHNR株的增殖特性、致病力及遗传稳定性等生物学特性进行检测，并通过接种2周龄SPF鸡评估rY2-FHNR株的免疫原性及其免疫血清与Y2株的交叉反应性，利用NDV NP蛋白的间接ELISA方法对免疫血清进行检测，验证其鉴别诊断效果。【结果】试验成功获得了嵌合重组病毒rY2-FHNR株，生物学特性检测结果显示，rY2-FHNR株在鸡胚中的增殖滴度和致病性符合弱毒特征，其鸡胚传代的遗传稳定性良好。rY2-FHNR株可诱导机体产生针对NDV的抗体，且免疫血清不与rY2株抗原发生交叉反应。通过NDV NP ELISA抗体检测方法可以实现区分疫苗免疫与野毒感染。【结论】本试验研发了一种基因Ⅶ型NDV嵌合候选疫苗，为基因Ⅶ型NDV的监测、防控和净化提供了技术支撑。

关键词: 新城疫病毒(NDV); 基因Ⅶ型; 禽偏禽腮腺炎病毒血清2型(AMAV-2); 反向遗传技术; 嵌合疫苗

Abstract: 【Objective】 This study was aimed to construct a genotype Ⅶ Newcastle disease virus (NDV) chimeric vaccine, and access its immune efficacy.【Method】 The F and HN protein ectodomains of recombinant plasmid pT7-Y2 of the Avian metaavulavirus-2 (AMAV-2) Y2 strain were replaced with the corresponding F and HN protein ectodomains of genotype Ⅶ NDV HB0901 strain by utilizing reverse genetic technology.Then F protein cleavage site of HB0901 strain was transformed into that of lentogenic LaSota strain, and the chimeric virus was recovered and named rY2-FHNR.The growth characteristics, pathogenicity and genetic stability of rY2-FHNR strain were evaluated, then 2-week-old SPF chickens were immunized with rY2-FHNR to determine the immunogenicity and cross-reactivity between immune serum and Y2 strain.The immune serum was identified by indirect NDV NP ELISA to verify its diagnosis effect.【Result】 Chimeric recombinant virus strain rY2-FHNR was constructed successfully.The biological characteristics results showed that the proliferation titer and pathogenicity in chicken embryos of rY2-FHNR strain were avirulent, while the chimeric virus was genetic stable when serially passaged in chicken embryos.Immunization with rY2-FHNR stimulated antibodies against NDV, and the immune serum had no cross-reactivity with rY2 strain.Through indirect NDV NP ELISA could distinguish infected animals from vaccinated animals.【Conclusion】 This study developed a genotype Ⅶ NDV chimeric vaccine candidate, which could be used as chimeric vaccine, provided technological support for the monitoring, prevention and eradication of genotype Ⅶ NDV.

Key words: Newcastle disease virus (NDV); genotype Ⅶ; Avian metaavulavirus-2(AMAV-2); reverse genetic technology; chimeric vaccine

中图分类号:

S852.65⁺7

李丽, 王国康, 商雨, 徐英英, 冯贺龙, 汪宏才, 张蓉蓉, 曾哲, 罗青平, 邵华斌, 曾驰, 温国元. 基于F和HN蛋白的基因Ⅶ型新城疫病毒嵌合疫苗的构建及免疫效力评估[J]. 中国畜牧兽医, 2022, 49(11): 4346-4355.

LI Li, WANG Guokang, SHANG Yu, XU Yingying, FENG Helong, WANG Hongcai, ZHANG Rongrong, ZENG Zhe, LUO Qingping, SHAO Huabin, ZENG Chi, WEN Guoyuan. Construction and Immune Efficacy Evaluation of a Genotype Ⅶ Newcastle Disease Virus Chimeric Vaccine Based on F and HN Proteins[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4346-4355.

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基于F和HN蛋白的基因Ⅶ型新城疫病毒嵌合疫苗的构建及免疫效力评估

Construction and Immune Efficacy Evaluation of a Genotype Ⅶ Newcastle Disease Virus Chimeric Vaccine Based on F and HN Proteins

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