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

作　　者：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[1] 张克山[2] 郑海学[2] 刘湘涛[2] 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(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;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)

机构地区：[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

出　　处：《畜牧兽医学报》2021年第6期1652-1661,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(NSFC-31972684);甘肃省自然科学基金(面上项目20JR5RA582)。

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。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.

关 键 词：组织蛋白酶S FMDV-O PK-15细胞 抗病毒功能 

分 类 号：S852.659.6[农业科学—基础兽医学]