猪源组织蛋白酶S抑制O型口蹄疫病毒在PK-15细胞复制

doi:10.11843/j.issn.0366-6964.2021.06.019

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (6): 1652-1661.doi: 10.11843/j.issn.0366-6964.2021.06.019

猪源组织蛋白酶S抑制O型口蹄疫病毒在PK-15细胞复制

史喜绢^1,2, 刘原子², 张大俊², 侯景², 申超超², 杨博², 张婷², 袁兴国¹, 任瑞瑞², 杜晓华^1*, 张克山^2*, 郑海学², 刘湘涛²

1. 甘肃农业大学动物医学院, 兰州 730070;
2. 中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室国家口蹄疫参考实验室, 兰州 730046

收稿日期:2020-09-09 出版日期:2021-06-23 发布日期:2021-06-22
通讯作者: 杜晓华,主要从事动物解剖学与组织学研究,E-mail:duxh@gsau.edu.cn;张克山,主要从事兽医微生物及其分子生物学研究,E-mail:zks009@126.com
作者简介:史喜绢(1994-),女,甘肃平凉人,硕士生,主要从事兽医病毒学研究,E-mail:shixijuan103@163.com
基金资助:
国家自然科学基金（NSFC-31972684）；甘肃省自然科学基金（面上项目20JR5RA582）

Porcine Cathepsin S Inhibits the Replication of Foot-and-Mouth Disease Virus Serotype O in PK-15 Cells

SHI Xijuan^1,2, LIU Yuanzi², ZHANG Dajun², HOU Jing², SHEN Chaochao², YANG Bo², ZHANG Ting², YUAN Xingguo¹, REN Ruirui², DU Xiaohua^1*, ZHANG Keshan^2*, ZHENG Haixue², LIU Xiangtao²

1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
2. State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Animal Virology of Ministry of Agriculture/National Foot-and-Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences, Lanzhou 730046, China

Received:2020-09-09 Online:2021-06-23 Published:2021-06-22

摘要/Abstract

摘要： 前期研究数据表明组织蛋白酶S（cathepsin S，CTSS）在猪初乳中表达水平显著高于常乳，且CTSS有抑制病毒复制的作用，本研究旨在探究猪源CTSS对O型口蹄疫病毒（foot-and-mouth disease virus serotype O，FMDV-O）复制及对FMDV诱导的抗病毒细胞因子的影响。FMDV-O感染PK-15细胞，利用RT-qPCR和Western blot分别在转录和翻译水平探究FMDV-O感染对内源性CTSS表达的影响；使用CTSS活性检测试剂盒测定FMDV-O感染对CTSS酶活性的影响；根据GenBank公布的CTSS基因序列（XM_021089893.1）构建CTSS真核表达质粒，利用脂质体方法转染PK-15细胞，通过Western blot检测CTSS表达情况，并在此基础上通过Western blot和RT-qPCR检测过表达CTSS对FMDV-O复制及FMDV-O诱导的抗病毒细胞因子mRNA水平的影响；进一步针对CTSS设计合成3对特异性siRNA，利用Western blot和RT-qPCR检测CTSS和FMDV-O的变化。结果表明，FMDV-O感染PK-15细胞能显著上调猪源CTSS表达并增强CTSS酶活性；过表达CTSS能抑制FMDV-O在PK-15细胞中复制，这种抑制作用可能是通过促进FMDV-O诱导的抗病毒细胞因子产生而发挥功能的；干扰序列siRNA-2947下调内源性CTSS表达进而促进FMDV-O的复制。猪源CTSS促进宿主抗病毒细胞因子产生可能是抑制FMDV-O复制的原因之一，本研究为深入探究宿主CTSS在抗FMDV天然免疫应答中的作用及机制提供依据。

关键词: 组织蛋白酶S, FMDV-O, PK-15细胞, 抗病毒功能

Abstract: The previous research data showed that the expression level of cathepsin S (CTSS) in sow colostrum was significantly higher than that of mature milk and CTSS has the effect of inhibiting virus replication. This study aims to explore the effect of pig-derived CTSS on the replication of foot-and-mouth disease virus serotype O (FMDV-O) and the production of antiviral cytokines induced by FMDV-O.PK-15 cells were infected with FMDV-O. The effect of FMDV-O infection on the expression of endogenous CTSS was investigated by RT-qPCR and Western blot at the transcriptional and translational level, respectively, and the effect of FMDV-O infection on the enzyme activity of CTSS in vitro was determined by CTSS activity detection kit. According to the CTSS gene sequence (XM_021089893.1) published by GenBank, the eukaryotic expression plasmid of CTSS was constructed and transfected into PK-15 cells by liposome. The expression of CTSS was detected by Western blot. On this basis, the effect of overexpressed CTSS on FMDV-O replication and the level of antiviral cytokine mRNA induced by FMDV-O was detected by Western blot and RT-qPCR, and three pairs of siRNA specific to CTSS were designed and synthesized. Western blot and RT-qPCR were used to detect the changes of CTSS and FMDV-O. The results showed that PK-15 cells infected with FMDV-O could significantly up-regulate the expression of porcine CTSS and enhance the activity of CTSS enzyme; overexpression of CTSS could inhibit the replication of FMDV-O in PK-15 cells, which may be through promoting the production of antiviral cytokines induced by FMDV-O; the interference sequence siRNA-2947 down-regulated the expression of endogenous CTSS and promoted the replication of FMDV-O. Porcine CTSS promoting the production of host antiviral cytokines may be one of the reasons for inhibiting FMDV-O replication. This study provides a basis for further exploring the role and mechanism of host CTSS in anti-FMDV innate immune response.

Key words: cathepsin S, FMDV-O, PK-15 cell, antiviral function

中图分类号:

S852.659.6

史喜绢, 刘原子, 张大俊, 侯景, 申超超, 杨博, 张婷, 袁兴国, 任瑞瑞, 杜晓华, 张克山, 郑海学, 刘湘涛. 猪源组织蛋白酶S抑制O型口蹄疫病毒在PK-15细胞复制[J]. 畜牧兽医学报, 2021, 52(6): 1652-1661.

SHI Xijuan, LIU Yuanzi, ZHANG Dajun, HOU Jing, SHEN Chaochao, YANG Bo, ZHANG Ting, YUAN Xingguo, REN Ruirui, DU Xiaohua, ZHANG Keshan, ZHENG Haixue, LIU Xiangtao. Porcine Cathepsin S Inhibits the Replication of Foot-and-Mouth Disease Virus Serotype O in PK-15 Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(6): 1652-1661.

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猪源组织蛋白酶S抑制O型口蹄疫病毒在PK-15细胞复制

Porcine Cathepsin S Inhibits the Replication of Foot-and-Mouth Disease Virus Serotype O in PK-15 Cells

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