与新城疫病毒NP蛋白互作宿主细胞蛋白的筛选和鉴定

doi:10.11843/j.issn.0366-6964.2025.08.032

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (8): 3942-3957.doi: 10.11843/j.issn.0366-6964.2025.08.032

与新城疫病毒NP蛋白互作宿主细胞蛋白的筛选和鉴定

韩银¹^,²(), 谢紫葳¹^,², 严舒可¹, 徐飞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:2024-10-18 出版日期:2025-08-23 发布日期:2025-08-28
通讯作者: 陈瑞爱 E-mail:hanyin928@163.com;chensa727@vip.126.com
作者简介:韩银(2000-)，男，湖北黄冈人，硕士生，主要从事畜禽疫病防控研究, E-mail: hanyin928@163.com
基金资助:
广东省重点领域研发计划项目(2021B0707010009)；岭南现代农业科学与技术广东省实验室肇庆分中心自主立项项目(P20211154-0301)

Screening and Identification of Host Proteins Interacting with NP of Newcastle Disease Virus

HAN Yin¹^,²(), XIE Ziwei¹^,², YAN Shuke¹, XU Fei¹^,², CHEN Ruiai¹^,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:2024-10-18 Online:2025-08-23 Published:2025-08-28
Contact: CHEN Ruiai E-mail:hanyin928@163.com;chensa727@vip.126.com

摘要/Abstract

摘要：

新城疫是由新城疫病毒(Newcastle disease virus，NDV)强毒株引起的一种急性接触性传染病，临床上常导致呼吸系统和消化系统的临床症状，NDV的广泛流行给家禽养殖业造成了巨大经济损失。NP(nucleocapsid)是NDV的一种核衣壳蛋白，在病毒复制、介导免疫应答和引发细胞自噬方面具有重要作用，其是否存在其他相互作用的宿主蛋白尚不清楚。为了筛选与NDV NP互作的宿主蛋白，初步探索互作宿主蛋白对NDV复制的影响，为NDV抗病药物新靶点的筛选提供理论基础。利用真核表达载体pXJ40成功构建了NDV NP的真核表达质粒，通过免疫共沉淀、质谱分析、GST-pull down以及激光共聚焦等技术筛选出能与NP相互作用的宿主蛋白，进一步在DF-1细胞中通过敲低、过表达等方法探究互作宿主蛋白对NDV复制的影响。质谱结果筛选到与NP发生相互作用的潜在蛋白211个，对差异蛋白进行富集分析揭示了其可能发挥的生物学功能和参与的生物学过程，进一步验证了热休克蛋白家族成员70(HSP70)与NP相互作用，并且这种互作是由HSP70的SBD结构域介导的；NDV感染能下调宿主细胞内HSP70的表达；过表达HSP70能在蛋白和转录水平显著抑制病毒复制；敲低内源性HSP70和HSP70抑制剂均能显著促进NDV复制。本研究筛选出NDV NP的互作宿主蛋白HSP70，并且验证了过表达HSP70能显著抑制NDV复制，作为NDV感染的负调控因子，为设计以HSP70为靶点的抗病毒药物提供了新思路。

关键词: 新城疫病毒, NP, HSP70蛋白, 免疫共沉淀技术

Abstract:

Newcastle disease is an acute contact infectious disease caused by Newcastle disease virus (NDV), which is characterized by respiratory and digestive symptoms. The widespread epidemic of NDV has caused huge economic losses to the poultry industry. NP is a nucleocapsid protein of NDV, which plays an important role in viral replication, immune response and cellular autophagy, while the host proteins interacting with NP remains unclear. The study was aim to screen the host proteins interacting with NDV NP and explore the effects of the host proteins on the replication of NDV, as well as to provide a theoretical basis for disease-resistant drug development. The eukaryotic expression plasmid of NDV NP was successfully constructed with the eukaryotic expression vector pXJ40. The host proteins interacting with NDV NP were screened by co-immunoprecipitation, mass spectrometry, GST-pull down, and laser confocal microscopy. The effects of the host proteins on NDV replication were explored by overexpression and knockdown in DF1cells. A total of 211 potential host proteins interacting with NP were screened by mass spectrometry. Enrichment analysis of the differential proteins revealed their possible biological functions and involvement in biological processes. Further detailed analysis of the interaction between HSP70 and NP confirmed the direct interaction of the two proteins and mapped the domain responsible for this binding to the SBD region in HSP70. NDV infection down-regulates HSP70 expression in host cells. Overexpression of HSP70 significantly inhibited viral replication at both the protein and transcriptional levels. However, both knockdown of endogenous HSP70 and use of HSP70 inhibitors significantly promoted NDV replication. This study indicated that the host protein HSP70 interacts with NDV NP and the overexpression of HSP70 could significantly inhibit NDV replication and act as a negative regulator of NDV infection, which provided a new idea for the design of antiviral drug targeting HSP70.

Key words: Newcastle disease virus, NP, HSP70 protein, co-immunoprecipitation

中图分类号:

S852.659.5

韩银, 谢紫葳, 严舒可, 徐飞, 陈瑞爱. 与新城疫病毒NP蛋白互作宿主细胞蛋白的筛选和鉴定[J]. 畜牧兽医学报, 2025, 56(8): 3942-3957.

HAN Yin, XIE Ziwei, YAN Shuke, XU Fei, CHEN Ruiai. Screening and Identification of Host Proteins Interacting with NP of Newcastle Disease Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3942-3957.

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引物名称 Primer name	序列 Primer sequences
pXJ40-Flag-NP-F	5′-gataagtccggatccaagcttATGTCGTCTGTTTTTGACGAATACG-3′
pXJ40-Flag-NP-R	5′-agttctgctttaataagatctTCAGTACCCCCAGTCAGTGTCG-3′
pXJ40-Myc-HSP70-F	5′-gaagaggatctgtccggatccATGTCTGGCAAAGGGCCG-3′
pXJ40-Myc-HSP70-R	5′-tctggtaccgagctcctgcagTTAATCTACTTCTTCAATGGTTGGGC-3′
pXJ40-Myc-HSP70-NBD-F	5′-gaagaggatctgtccggatccGGCAAAGGGCCGGCCATC-3′
pXJ40-Myc-HSP70-NBD-R	5′-tctggtaccgagctcctgcagTTCAGACTTGTCTCCCATGAGGA-3′
pXJ40-Myc-HSP70-SBD-F	5′-gaagaggatctgtccggatccTTGGATGTCACCCCCCTGT-3′
pXJ40-Myc-HSP70-SBD-R	5′-tctggtaccgagctcctgcagGCGACCCTTGTCATTGGTGA-3′
pXJ40-Flag-F	5′-ACATCCTGGTCATCATCCTG-3′
pXJ40-Flag-R	5′-ACCACAACTAGAATGCAGTG-3′
pXJ40-Myc-F	5′-ATCCTGCCTTTCTCTTTATGGT-3′
pXJ40-Myc-R	5′-TAACCGTATTACCGCCTTTGAGTG-3′

引物名称 Primer name	引物序列 Primer sequences
q-DHN3-F	5′-CTCATAGCAGGCTCCTGTCG-3′
q-DHN3-R	5′-CAGTTGTGCGATCATGCGAG-3′
q-NP-F	5′-TGAGCACATCATTCTGGCGA-3′
q-NP-R	5′-CAGATACTCGTTGGGCAGCA-3′
q-HSP70-F	5′-ATTCTTGCGTGGGTGTCTTC-3′
q-HSP70-R	5′-GATGGTGTTGGTGGGGTTC-3′
q-β-Actin-F	5′-CTGTGCCCATCTATGAAGGCTA-3′
q-β-Actin-R	5′-ATTTCTCTCTCGGCTGTGGTG-3′

名称Name	靶序列Target sequence
si-HSP70 1#	CCCGCTTACTTCAACGACT
si-HSP70 2#	CAAGAAGCAGAGAAATACA
si-HSP70 3#	CAAGAAGCAGAGAAATACA

登录号 Accession No.	基因名称 Gene Name	大小/ku MW	覆盖率/% Coverage	肽段 Peptides
A0A1C9KD18	ANXA2	38.6	71	27
Q7SX63	HSP70	69.9	49	31
H9KZT7	ILF2	54.3	3	1
F1NJS6	KPNA2	55.9	28	10
F1NVA4	NPM1	43.7	28	10
A0A1D5NYB2	PABPC1	70.8	36	23
A0A1D5PJW4	PSMA7	27.9	44	10
Q6DV79	STAT3	88.1	14	7

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与新城疫病毒NP蛋白互作宿主细胞蛋白的筛选和鉴定

Screening and Identification of Host Proteins Interacting with NP of Newcastle Disease Virus

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