犬ANP32蛋白多态性及其对A型流感病毒RNA聚合酶活性的影响

doi:10.16431/j.cnki.1671-7236.2022.07.004

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (7): 2462-2473.doi: 10.16431/j.cnki.1671-7236.2022.07.004

犬ANP32蛋白多态性及其对A型流感病毒RNA聚合酶活性的影响

张媛¹, 于萌萌¹, 孙留克¹, 郭兴¹, 那雷¹, 刘荻萩¹, 姜骞¹, 张海丽^1,2, 王晓钧¹

1. 中国农业科学院哈尔滨兽医研究所, 兽医生物技术国家重点实验室, 哈尔滨 150069;
2. 吉林大学动物医学学院, 人兽共患病研究教育部重点实验室, 长春 130062

收稿日期:2021-11-18 出版日期:2022-07-05 发布日期:2022-06-29
通讯作者: 张海丽, 王晓钧 E-mail:zhanghaili@jlu.edu.cn;wangxiaojun@caas.cn
作者简介:张媛,E-mail:zhangyuan19950804@163.com。
基金资助:
国家自然科学基金青年科学基金项目（32002275）；黑龙江省自然科学基金优秀青年项目（YQ2020C021）

Analysis of Canine ANP32 Protein Polymorphism and Effect on RNA Polymerase Activity of Influenza A Virus

ZHANG Yuan¹, YU Mengmeng¹, SUN Liuke¹, GUO Xing¹, NA Lei¹, LIU Diqiu¹, JIANG Qian¹, ZHANG Haili^1,2, WANG Xiaojun¹

1. State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China;
2. Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, China

Received:2021-11-18 Online:2022-07-05 Published:2022-06-29

摘要/Abstract

摘要： 【目的】探索犬酸性核磷蛋白32（canis acidic （leucine-rich） nuclear phosphoprotein 32 ku，caANP32）对不同物种A型流感病毒（Influenza A virus，IAV） RNA 聚合酶活性的影响。【方法】利用实时荧光定量PCR方法分析caANP32家族基因（caANP32A、caANP32B及caANP32E）的组织分布并对其进行扩增和克隆，评估caANP32家族蛋白对不同物种A型流感病毒RNA聚合酶活性的支持作用。【结果】caANP32家族基因在不同组织中分布相似，其中caANP32B基因组织丰度显著或极显著高于caANP32A和caANP32E基因（P＜0.05；P＜0.01），在心脏、盲肠和大脑中caANP32E基因组织丰度极显著或显著高于caANP32A基因（P＜0.01；P＜0.05），在肝脏、肺脏等组织中caANP32E与caANP32A基因丰度均无显著差异（P>0.05）。在犬心脏组织和MDCK细胞中发现，caANP32A基因仅存在1个转录本，caANP32B基因存在3个不同剪接变体：caANP32B_X1、caANP32B_X2和caANP32B_X3；caANP32E 基因具有2个不同剪接变体：caANP32E_X1和caANP32E_X2。通过分析ANP32家族蛋白对不同物种A型流感病毒RNA聚合酶活性的影响发现，caANP32A和caANP32B均能支持犬流感病毒（H3N2_GD11）和马流感病毒（H3N8_JL89）RNA聚合酶活性，而对禽流感病毒（H9N2_ZJ12）RNA聚合酶活性支持较低。caANP32B_X2剪接变体对哺乳动物流感病毒RNA聚合酶活性的支持能力显著高于caANP32B_X1和caANP32B_X3（P＜0.05）；而caANP32E不支持A型流感病毒RNA聚合酶活性。【结论】本研究初步解析了caANP32家族蛋白的组织分布及序列多态性，阐明了其对不同物种A型流感病毒RNA聚合酶活性的支持作用，为解析MDCK细胞作为流感病毒分离和疫苗生产细胞系的分子机制提供了新的借鉴。

关键词: A型流感病毒(IAV); 犬酸性核磷蛋白32(caANP32); MDCK细胞; 聚合酶活性

Abstract: 【Objective】 This study was aimed to explore the effect of canine acidic (leucine-rich) nuclear phosphoprotein 32 ku (caANP32) on the RNA polymerase activity of Influenza A virus (IAV) from different species.【Method】 Real-time PCR method was used to analyze the tissue distribution of caANP32 family genes (caANP32A,caANP32B and caANP32E),then these genes were amplified and cloned to evaluate the supporting effect of caANP32 family proteins on the RNA polymerase activity of IAV in different species.【Result】 The results showed that the caANP32 family genes were similarly expressed in different tissues,and the expression abundance of caANP32B gene was significantly or extremely significantly higher than that of caANP32A and caANP32E genes in different tissues (P＜0.05;P＜0.01),the tissue abundance of caANP32E gene was extremely significantly or significantly higher than that of caANP32A gene in heart,cecum and brain (P＜0.01;P＜0.05),there was no significant difference in gene abundance between caANP32E and caANP32A genes in liver,lung and other tissues (P>0.05).Only one transcript of caANP32A gene was found in canine heart tissue and MDCK cells,however three different splice variants of caANP32B gene (caANP32B_X1,caANP32B_X2 and caANP32B_X3), and two different splice variants of the caANP32E gene (caANP32E_X1 and caANP32E_X2) were found in these samples.By analyzing the effects of ANP32 family members on the RNA polymerase activity of IAV from different species,it was found that caANP32A and caANP32B could support Canine influenza virus (H3N2_GD11) and Equine influenza virus (H3N8_JL89) RNA polymerase activity,but had lower activity to support Avian influenza virus (H9N2_ZJ12) RNA polymerase activity.The supporting ability of caANP32B_X2 to the RNA polymerase activity of IAV was significantly higher than that of caANP32B_X1 and caANP32B_X3,while caANP32E did not support the RNA polymerase activity of Avian influenza virus.【Conclusion】 This study analyzed the tissue distribution and sequence polymorphisms of the caANP32 family members,and clarified their role in supporting RNA polymerase activity of IAV from different species,providing a new basis for analyzing the molecular mechanism of MDCK as a cell line for influenza virus isolation and vaccine production.

Key words: Influenza A virus (IAV); caANP32; MDCK cells; polymerase activity

中图分类号:

S852.65

张媛, 于萌萌, 孙留克, 郭兴, 那雷, 刘荻萩, 姜骞, 张海丽, 王晓钧. 犬ANP32蛋白多态性及其对A型流感病毒RNA聚合酶活性的影响[J]. 中国畜牧兽医, 2022, 49(7): 2462-2473.

ZHANG Yuan, YU Mengmeng, SUN Liuke, GUO Xing, NA Lei, LIU Diqiu, JIANG Qian, ZHANG Haili, WANG Xiaojun. Analysis of Canine ANP32 Protein Polymorphism and Effect on RNA Polymerase Activity of Influenza A Virus[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2462-2473.

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犬ANP32蛋白多态性及其对A型流感病毒RNA聚合酶活性的影响

Analysis of Canine ANP32 Protein Polymorphism and Effect on RNA Polymerase Activity of Influenza A Virus

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