新的犬ANP32A的克隆及其在流感病毒跨物种感染中的作用

doi:10.11843/j.issn.0366-6964.2024.02.023

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (2): 660-669.doi: 10.11843/j.issn.0366-6964.2024.02.023

新的犬ANP32A的克隆及其在流感病毒跨物种感染中的作用

毕振威^1,3*, 王文杰¹, 刘雅坤¹, 彭大新²

1. 江苏省农业科学院兽医研究所农业农村部兽用生物制品工程技术重点实验室, 南京 210014;
2. 扬州大学江苏省人兽共患病学重点实验室, 扬州 225009;
3. 兽用生物制品(泰州)国泰技术创新中心, 泰州 225300

收稿日期:2023-06-16 出版日期:2024-02-23 发布日期:2024-02-27
通讯作者: 毕振威,E-mail:bizhenwei@126.com;Tel:025-84390339
作者简介:毕振威(1985-),男,山东威海人,副研究员,博士,主要从事动物病毒跨物种感染传播机制研究
基金资助:
江苏省人兽共患病学重点实验室资助项目(R2101)

Cloning of a New Canine ANP32A and Its Role in Cross-species Infection of Influenza Virus

BI Zhenwei^1,3*, WANG Wenjie¹, LIU Yakun¹, PENG Daxin²

1. Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture and Rural Affsirs, Nanjing 210014, China;
2. Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou 225009, China;
3. GuoTai(Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China

Received:2023-06-16 Online:2024-02-23 Published:2024-02-27

摘要/Abstract

摘要： 物种特异的酸性核磷蛋白32A(acidic nuclear phosphoprotein 32A, ANP32A)调节不同宿主A型流感病毒RNA聚合酶活性。为分析犬ANP32A(canine ANP32A, caANP32A)的物种特异性及其在流感病毒跨物种感染中的作用,利用RT-PCR方法从MDCK细胞以及犬肺、脾、肠不同组织中扩增和克隆caANP32A;激光共聚焦试验分析caANP32A与A型流感病毒RNA聚合酶的相互作用;双荧光素酶报告基因试验检测过表达caANP32A对A型流感病毒RNA聚合酶活性的影响。结果显示,从MDCK细胞中扩增到新的caANP32A,比已报道的caANP32A多出4个氨基酸插入,从犬肺、脾和肠组织中均扩增到该新的caANP32A;对caANP32A的基因进行测序分析,发现该新的caANP32A不是由mRNA选择性剪接形成的;激光共聚焦试验发现,新caANP32A与H3N2 CIV的RNA聚合酶在细胞核中共定位;聚合酶活性试验显示,在哺乳动物细胞过表达新caANP32A不能促进H9N2禽流感病毒(avian influenza virus,AIV)和H3N2犬流感病毒(canine influenza virus, CIV)的RNA聚合酶活性,而鸡ANP32A(chANP32A)能够促进。本研究克隆的新caANP32A较以往报道,在176至179位存在四个氨基酸LSLV的插入,但新caANP32A对AIV的RNA聚合酶活性仍然有物种限制性且该新的caANP32A也未增强CIV的RNA聚合酶活性。本研究为进一步解析犬在流感病毒跨物种感染中的作用提供依据。

关键词: A型流感病毒(IAV), 犬酸性核磷蛋白32A(caANP32A), RNA聚合酶活性, 哺乳动物适应性

Abstract: The species-specific acidic nuclear phosphoprotein 32A (ANP32A) regulates RNA polymerase activity of influenza A virus from different hosts. To analyze species-specific difference of canine ANP32A (canine ANP32A, caANP32A) and its role in the cross-species infection of influenza virus, RT-PCR was used to amplify and clone caANP32A from Madin-Darby canine kidney (MDCK) cells and different tissues of canine lung, spleen and intestine. The interaction of caANP32A with influenza A virus RNA polymerase was analyzed by Laser confocal assay. The effect of caANP32A on RNA polymerase activity of influenza A virus was detected in 293T cells by double luciferase reporter gene experiment. The results showed that the new caANP32A was amplified from MDCK cells, which had four more amino acids insertion than the previously reported caANP32A. The new caANP32A was amplified from the lung, spleen and intestine of dogs; After sequencing and analyzing the ANP32A gene, it was found that the new caANP32A was not formed by alternative splicing of mRNA; Laser confocal assay showed that the co-location of the new caANP32A with the RNA polymerase of H3N2 CIV in the nucleus. Polymerase activity experiments showed that overexpression of novel caANP32A in mammalian cells did not promote the RNA polymerase activity of H9N2 avian influenza virus (AIV) and H3N2 canine influenza virus (CIV), but chANP32A could. The new caANP32A cloned in this study, compared to previous reports, has four amino acids LSLV insertion at positions 176 to 179, but the new caANP32A still has species restriction on the polymerase activity of AIV and does not enhance the RNA polymerase activity of CIV. This study provides a basis for further elucidating the role of dogs in the cross-species infection of influenza virus.

Key words: influenza A virus (IAV), canine acidic nuclear phosphoprotein 32 (caANP32A), RNA polymerase activity, mammalian adaptability

中图分类号:

S852.659.5

毕振威, 王文杰, 刘雅坤, 彭大新. 新的犬ANP32A的克隆及其在流感病毒跨物种感染中的作用[J]. 畜牧兽医学报, 2024, 55(2): 660-669.

BI Zhenwei, WANG Wenjie, LIU Yakun, PENG Daxin. Cloning of a New Canine ANP32A and Its Role in Cross-species Infection of Influenza Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 660-669.

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新的犬ANP32A的克隆及其在流感病毒跨物种感染中的作用

Cloning of a New Canine ANP32A and Its Role in Cross-species Infection of Influenza Virus

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