猪TRIM56敲除细胞系的构建及在增殖PRRSV疫苗毒株中的应用  

Construction of swine TRIM56 knockout cell line and its application in propagation of PRRSV vaccine strain

作　　者：张秀婷 吴香菊 齐静[2] 丛晓燕[2] 李均同[2] 陈大全[1] 姜宁朋 杜以军[2] ZHANG Xiuting;WU Xiangju;QI jing;CONG Xiaoyan;LI Juntong;CHEN Daquan;JIANG Ningpeng;DU Yijun(School of Pharmacy,Yantai University,Yantai 264005,China;Shandong Province Key Laboratory of Animal Disease Control and Breeding/Institute of Animal Science and Veterinary Medicine,Shandong Academy of Agricultural Sciences,Jinan 250100,China;Shandong Science and Technology Service Development Promotion Center,Jinan 250101,China)

机构地区：[1]烟台大学药学院,山东烟台264005 [2]山东省农业科学院畜牧兽医研究所/山东省畜禽疫病防治与繁育重点实验室,山东济南250100 [3]山东省科技服务发展推进中心,山东济南250101

出　　处：《畜牧与兽医》2023年第11期117-123,共7页Animal Husbandry & Veterinary Medicine

基　　金：山东省自然科学基金项目(ZR2020QC196,ZR2020KC005,ZR2021MC139,ZR2021ZD08);国家自然科学基金项目(32102710);国家重点研发计划(2021YFD1800300);山东省农业科学院农业科技创新项目(CXGC2023A21)。

摘　　要：利用CRISPR/Cas9基因编辑技术构建1株猪TRIM56基因敲除的PAM-KNU细胞系,并探究其在增殖猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)疫苗毒株中的应用。根据猪TRIM56基因组外显子区域设计合成2对特定的向导RNA引物,将引物磷酸化、退火并连接至Px459M和EZ-Guide-XH载体构建敲除质粒Px459M-sTRIM56-KO;将敲除质粒转染PAM-KNU细胞,经嘌呤霉素筛选、有限稀释法筛选获得单克隆细胞并扩大培养;通过RT-PCR、测序及Western blot筛选鉴定,最终获得了敲除细胞系sTRIM56-KO-PAM-KNU;将PRRSV R98疫苗株感染野生型细胞及敲除细胞系,利用real-time RT-PCR、半数组织培养感染量(TCID_(50))以及Western blot检测PRRSV增殖情况。结果显示,TRIM56敲除细胞系构建成功,该敲除细胞系中sTRIM56基因编码区第929~2120位1192个碱基缺失,未检测到sTRIM56蛋白条带;敲除细胞系中PRRSV R98疫苗株病毒拷贝数、病毒滴度以及N蛋白表达均显著高于对照组。本研究为猪TRIM56抗病毒机制研究及PRRSV疫苗研发奠定了基础。The aim of this study was to construct a TRIM56-knockout immortalized porcine alveolar macrophage cell line PAM-KNU using the CRISPR/Cas9 gene editing technique and to explore the application of the cell line in the propagation of porcine reproductive and respira⁃tory syndrome virus(PRRSV)vaccine strains.Two pairs of specific guide RNA primers were designed and synthesized according to the exon⁃ic region of the swine TRIM56 gene.The primers were phosphorylated,annealed and ligated to Px459M and EZ-Guide-XH vectors to con⁃struct knockout plasmid Px459M-sTRIM56-KO.PAM-KNU cells were transfected with the knockout plasmid and were selected with puro⁃mycin.Monoclonal cells were obtained by limited dilution and were expanded in vitro.The cells were verified by RT-PCR,sequencing,Western blot and were named sTRIM56-KO-PAM-KNU.The wild type and knockout cell lines were infected with the PRRSV R98 vaccine strain.The PRRSV propagation was detected by real-time RT-PCR,TCID50 and Western blot.The results showed that a TRIM56 knockout cell line was established,1192 bases at positions 929~2120 were deleted in the coding region of the sTRIM56 gene,and the sTRIM56 pro⁃tein band was not detected.The virus copies,virus titer and expression of PRRSV N proteins in the knockout cell line were significantly high⁃er than those in the control cells.This study laid a foundation for the study of antiviral mechanism of swine TRIM56 and the development of PRRSV vaccines.

关 键 词：猪TRIM56基因 基因敲除 PAM-KNU细胞 猪繁殖与呼吸综合征病毒 

分 类 号：S852[农业科学—基础兽医学]