猪源IFN-λ3在CHO-S细胞中的表达及其抗口蹄疫病毒活性分析  

Expression of porcine IFN-λ3 in CHO-S cells and analysis of its antiviral activity against foot-and-mouth disease virus

作　　者：袁红[1] 白兴文[1] 李坤[1] 李平花[1] 孙普[1] 宋雅丽 焦云娟 刘在新[1] 卢曾军[1] 李冬[1] 包慧芳[1] YUAN Hong;BAI Xing-wen;LI Kun;LI Ping-hua;SUN Pu;SONG Ya-li;JIAO Yun-juan;LIU Zai-xin;LU Zeng-jun;LI Dong;BAO Hui-fang(State Key Laboratory of Veterinary Etiological Biology/National Foot-and-Mouth Disease Reference Laboratory/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]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国家口蹄疫参考实验室,甘肃兰州730046

出　　处：《中国兽医科学》2022年第2期190-196,共7页Chinese Veterinary Science

基　　金：宁夏回族自治区重点研发计划重大(重点)项目(2019BBF02005);国家自然科学基金面上项目(31772763)。

摘　　要：为了探究猪源IFN-λ3蛋白能否在CHO-S悬浮细胞中表达以及表达蛋白的抗口蹄疫病毒(FMDV)活性,根据NCBI上的猪源IFN-λ3序列,构建真核表达载体pcDNA3.4-IFNλ3-His,将其转染至CHO-K1贴壁细胞中,用间接免疫荧光试验和Western-blot验证重组质粒是否表达IFN-λ3蛋白。将pcDNA3.4-IFNλ3-His转染至CHO-S悬浮细胞中大量制备IFN-λ3蛋白,用AKTA蛋白纯化系统纯化蛋白,用SDS-PAGE和Western-blot方法鉴定纯化出的蛋白,用细胞毒性试验分析纯化蛋白是否具有细胞毒性,用实时荧光定量PCR、Western-blot和空斑试验三种方法研究纯化蛋白的抗FMDV活性。结果显示,重组质粒pcDNA3.4-IFNλ3-His在CHO-K1细胞中瞬时表达IFN-λ3,在CHO-S细胞中以分泌形式表达IFN-λ3;用AKTA系统能够纯化出纯度较高的IFN-λ3蛋白;纯化出的IFN-λ3对PK-15细胞无明显毒性,能够抑制FMDV RNA的复制、减弱病毒蛋白翻译活动以及降低子代病毒的生成。本研究为发展新型FMDV防控策略提供了新方向。In order to explore whether porcine IFN-λ3 protein could be expressed in CHO-S suspension cells and whether the expressed porcine IFN-λ3 had antiviral activities against foot-and-mouth disease virus(FMDV),the eukaryotic expression plasmid pc DNA3.4-IFNλ3-His was constructed based on porcine IFN-λ3 sequence on NCBI.Then the recombinant plasmid was transfected into CHO-K1 cells,and the expression of IFN-λ3 protein was identified by indirect immunofluorescence test and Western-blot.Subsequently,porcine IFN-λ3 protein was prepared in large quantities through transfection of pc DNA3.4-IFNλ3-His into CHO-S suspension cells,and purified by AKTA system.The purified protein was analyzed through SDS-PAGE and Western-blot analysis.Moreover,the cytotoxicity of purified protein was determined by cell viability assay,and antiviral activities against FMDV were measured using reverse transcription quantitative real-time PCR(RT-q PCR),Western-blot and plaque assay.The data showed that recombinant plasmid pc DNA3.4-IFNλ3-His was able to transiently express IFN-λ3 in CHO-K1 cells and also in the supernatant of CHO-S cells in a secreted form.The expressed IFN-λ3 could be purified by AKTA system with higher purity.In addition,we found that the purified IFN-λ3 showed no apparent cytotoxicity to PK-15 cells and significantly inhibited FMDV RNA replication,weakened viral protein translation and reduced the generation of progeny viruses.This research provided a new direction for the development of new prevention and control strategies for FMDV.

关 键 词：猪源IFN-λ3 表达 CHO-S细胞 口蹄疫病毒 抗病毒活性 

分 类 号：S852.659.6[农业科学—基础兽医学]