共表达膜结合型与可溶性H9N2亚型禽流感病毒HA蛋白的重组基因Ⅶ型新城疫病毒的构建及免疫效果评价

doi:10.11843/j.issn.0366-6964.2024.05.030

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (5): 2123-2134.doi: 10.11843/j.issn.0366-6964.2024.05.030

共表达膜结合型与可溶性H9N2亚型禽流感病毒HA蛋白的重组基因Ⅶ型新城疫病毒的构建及免疫效果评价

吕亚迪^1,2, 杨洁^1,2, 谢文婷^1,2, 徐婷^1,2, 陈瑞爱^1,2,3,4*

1. 华南农业大学兽医学院, 广州 510642;
2. 岭南现代农业科学与技术广东省实验室肇庆分中心, 肇庆 526238;
3. 华农(肇庆)生物产业技术研究院有限公司, 肇庆 526238;
4. 肇庆大华农生物药品有限公司, 肇庆 526238

收稿日期:2023-08-10 出版日期:2024-05-23 发布日期:2024-05-27
通讯作者: 陈瑞爱，主要从事动物疾病防控科研等，E-mail:chensa727@vip.126.com
作者简介:吕亚迪(1998-)，女，河南商水人，硕士生，主要从事畜禽疫病防控研究，E-mail:15716327913@163.com；Tel：0758-3626781
基金资助:
广东省重点领域研发计划项目，鸡传染性支气管炎和禽流感病毒样颗粒疫苗的研制及应用(2021B0707010009)

Construction and Evaluation of the Immune Effect of Recombinant Genotype Ⅶ NDV Strain Co-expressing Membrane-bound and Water-soluble HA Protein of Avian Influenza Virus H9N2

LÜ Yadi^1,2, YANG Jie^1,2, XIE Wenting^1,2, XU Ting^1,2, CHEN Ruiai^1,2,3,4*

1. College of Veternary Medicine, South China Agricultural University, Guangzhou 510642, China;
2. Zhaoqing Branch Center of Guangdong Laboratory for Lingnan modern Agricultural Science and Technology Co., Ltd, Zhaoqing 526238, China;
3. Zhaoqing Institute of Biological Medicine Co., Ltd, Zhaoqing 526238, China;
4. Zhaoqing Dahuanong Biological Medicine Co., Ltd, Zhaoqing 526238, China

Received:2023-08-10 Online:2024-05-23 Published:2024-05-27

摘要/Abstract

摘要： 旨在构建能高效稳定表达H9N2 AIV的HA蛋白的重组基因Ⅶ型新城疫病毒，有望开发抗新城疫和H9N2亚型禽流感病毒的二联疫苗，为控制H9N2 AIV和NDV提供新的防御思路，对NDV疫苗载体的构建和优化有重要的参考意义。利用反向遗传技术，以rDHN3-mF为骨架，在病毒基因组的P和M之间的非编码区插入全长膜结合HA和另外一个带有截短跨膜结构域的可溶性HA蛋白，获得了能高效稳定表达H9N2 AIV的HA蛋白的基因Ⅶ型NDV减毒株重组病毒rDHN3mF-HA和rDHN3mF-2HA。通过鸡胚接种、Western blot、血凝效价(HA)及平均鸡胚致死时间(MDT)等试验来检测重组病毒的稳定性以及生物学特性，将重组病毒以10⁶ EID_50·只^-1剂量免疫7日龄SPF鸡并测定血凝抑制(HI)抗体，在免疫后28 d对各组进行攻毒保护试验。结果显示：重组病毒在鸡胚中连续传至十五代后HA基因无突变发生；Western blot证明重组病毒可稳定高效地表达特异性的HA蛋白；重组病毒的生长曲线说明了重组病毒与亲本毒株具有相似的复制特性和低毒力特征；重组病毒免疫组的SPF鸡均能产生针对NDV或者H9N2 AIV的特异性HI抗体；攻毒保护试验结果：重组病毒均能对攻毒后的鸡产生100%保护效果；喉、肛拭子检测结果说明重组病毒可显著减少NDV与H9N2 AIV的体外排毒；气管与肺qPCR检测结果显示攻毒后3 d，rDHN3mF-2HA较rDHN3mF-HA更显著地降低了脏器中H9N2 AIV含量。本研究成功构建了共表达膜结合型与可溶性H9N2亚型禽流感HA蛋白的基因Ⅶ型重组NDV，为H9N2 AIV和NDV的一种新型重组基因工程二联活载体疫苗的研制与应用奠定了基础。

关键词: H9N2亚型禽流感病毒, HA蛋白, 新城疫病毒, 免疫原性, 载体疫苗, 免疫保护效率

Abstract: The purpose of this study was to construct a recombinant genotype Ⅶ Newcastle disease virus (NDV) that can efficiently and stably express H9N2 subtype avian influenza virus (AIV) HA protein. It is expected to develop a bivalent vaccine against NDV and H9N2 AIV to provide a new defense idea for the control of H9N2 AIV and NDV, and has important reference significance for the construction and optimization of NDV vaccine vector. A reverse genetic technique was used to insert the full-length membrane-bound HA and another soluble HA protein with truncated transmembrane domain gene into the non-coding region between the P and M genes of NDV using the genotype Ⅶ NDV-weakening strain rDHN3-mF as the backbone. The recombinant virus rDHN3mF-HA and rDHN3mF-2HA of gene type VII NDV attenuated virus which can express HA protein stably and efficiently was obtained. The stability and biological characteristics of recombinant virus were detected by chicken embryo inoculation, Western blot, HA, MDT and other experiments. Recombinant virus was immunized with 7 d SPF chickens and serum HI assay were measured. And the group was subjected to challenge protection experiments 28 d after immunization. The recombinant viruses were passed through fifteen generations in chicken embryos without mutations.Western blot results demonstrated that recombinant viruses can express specific HA proteins stably and efficiently; The growth curves illustrates the recombinant viruses have similar replication and low virulence characteristics to parental strains; Groups of SPF chickens immunized with recombinant virus produce specific HI antibodies against NDV or H9N2 AIV.The challenge protection test showed that the recombinant viruses can produce 100% protection against chickens after challenge, and the results of throat and swab detection indicate that recombinant virus can significantly reduce the amount of NDV and H9N2 AIV shedding. The results of tracheal and lung qPCR showed that rDHN3mF-2HA significantly reduced the shedding of H9N2 AIV in organs than rDHN3mF-HA 3 dpc. The results indicated that the recombinant NDV of genotype Ⅶ coexpressing membrane-bound and soluble H9N2 subtype avian influenza HA protein was successfully constructed, which laid a foundation for the development and application of a new type of recombinant genetic engineering live vector vaccine to prevent H9N2 AIV and NDV.

Key words: H9N2 subtype avian influenza, HA protein, Newcastle disease virus, immunogenicity, vector vaccines, immunoprotective efficacy

中图分类号:

S858.315.3

吕亚迪, 杨洁, 谢文婷, 徐婷, 陈瑞爱. 共表达膜结合型与可溶性H9N2亚型禽流感病毒HA蛋白的重组基因Ⅶ型新城疫病毒的构建及免疫效果评价[J]. 畜牧兽医学报, 2024, 55(5): 2123-2134.

LÜ Yadi, YANG Jie, XIE Wenting, XU Ting, CHEN Ruiai. Construction and Evaluation of the Immune Effect of Recombinant Genotype Ⅶ NDV Strain Co-expressing Membrane-bound and Water-soluble HA Protein of Avian Influenza Virus H9N2[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2123-2134.

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共表达膜结合型与可溶性H9N2亚型禽流感病毒HA蛋白的重组基因Ⅶ型新城疫病毒的构建及免疫效果评价

Construction and Evaluation of the Immune Effect of Recombinant Genotype Ⅶ NDV Strain Co-expressing Membrane-bound and Water-soluble HA Protein of Avian Influenza Virus H9N2

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