过表达JMJD6基因的293T细胞系的构建及对水疱性口炎病毒增殖效率的评价  

Construction of 293T cell line overexpressing JMJD6 gene and evaluation of its incremental efficiency against vesicular stomatitis virus

作　　者：黄梦瑶 杨帆[1,2] 杨洋 邵文华[1,2] 王家丽 吕岳芹 赵晓义 陈治彤 曹伟军 张伟 郑海学[1,2] HUANG Mengyao;YANG Fan;YANG Yang;SHAO Wenhua;WANG Jiali;LU Yueqin;ZHAO Xiaoyi;CHEN Zhitong;CAO Weijun;ZHANG Wei;ZHENG Haixue(State Key Laboratory for Animal Disease Control and Prevention/College of Veterinary Medicine of Lanzhou University/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China;Gansu Province Research Center for Basic Disciplines of Pathogen Biology,Lanzhou 730046,China)

机构地区：[1]中国农业科学院兰州兽医研究所兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室,甘肃兰州730000 [2]甘肃省病原生物学基础学科研究中心,甘肃兰州730046

出　　处：《中国兽医科学》2024年第5期569-576,共8页Chinese Veterinary Science

基　　金：国家自然科学基金项目(32102639,32072831);国家重点研发计划项目(2021YFD1800300);甘肃省杰出青年基金项目(21JR7RA026);兰州大学中央高校基本科研业务费专项资金(lzujbky-2022-ey20);“十四五”广东省农业科技创新十大主攻方向“揭榜挂帅”项目(2023SDZG02);国家生猪产业技术体系(CARS-35)。

摘　　要：为提高水疱性口炎病毒(vesicular stomatitis virus,VSV)在细胞中的增殖效率,本研究利用慢病毒包装系统构建Jumonji C结构域蛋白6(Jumonji domain-containing protein 6,JMJD6)过表达的HEK-293T/JMJD6细胞系,分析VSV在HEK-293T/JMJD6细胞系与HEK-293T细胞中的复制差异。首先以JMJD6基因为目标,构建重组质粒pLV-puro-3×Flag-JMJD6,并与辅助质粒pMD2.G和psPAX2共转染至HEK-293T细胞进行慢病毒包装。将包装好的慢病毒感染HEK-293T细胞并通过嘌呤霉素筛选获得过表达JMJD6基因的阳性细胞。利用Western-blot、间接免疫荧光试验、流式细胞术和蚀斑试验检测VSV在HEK-293T/JMJD6细胞系中的复制能力。结果显示,在HEK-293T/JMJD6细胞系中,VSV的复制能力显著增强。实时荧光定量检测VSV感染的HEK-293T/JMJD6细胞系和野生型HEK-293T细胞中干扰素下游基因的表达,结果显示HEK-293T/JMJD6细胞系抑制VSV诱导的Ⅰ型干扰素下游基因的产生。总之,本研究构建的HEK-293T/JMJD6细胞系能显著促进VSV的增殖并抑制Ⅰ型干扰素信号通路,为VSV疫苗候选细胞株的筛选奠定了基础。In order to enhance the efficiency of vesicular stomatitis virus(VSV)proliferation within cells,this study employed a lentivirus packaging system to establish a HEK-293T/JMJD6 cell line that exhibited an overexpression of Jumonji domain containing protein 6(JMJD6).Subsequently,the replication disparities of VSV between HEK-293T/JMJD6 and HEK-293T cells were analyzed.To begin,a re Cloncombinant plasmid pLV-puro-3×Flag-JMJD6 gene was constructed and co-transfected with auxiliary plasmids pMD2.G and psPAX2 into HEK-293T cells for the purpose of lentivirus packaging.Following this,HEK-293T cells were infected with the packaged lentivirus,and positive cells overexpressing the JMJD6 gene were obtained by puromycin selection.To assess the replication ability of VSV in the HEK-293T/JMJD6 cell line,Western-blot,indirect immunofluorescence,flow cytometry,and plaque assay were employed.The results demonstrated that the proliferation ability of vesicular stomatitis virus(VSV)in the HEK-293T/JMJD6 cell line was significantly greater compared to wild-type cells.Reverse transcription polymerase chain reaction(RT-PCR)analysis of interferon downstream gene expression in VSV-infected HEK-293T/JMJD6 cell lines and wild-type cells revealed that the HEK-293T/JMJD6 cell line suppressed the production of type I interferon downstream genes induced by VSV.The establishment of the HEK-293T/JMJD6 cell line in this study effectively enhances VSV proliferation and inhibits the type I interferon signaling pathway,thereby providing a basis for the identification of potential VSV vaccine candidate cell lines.

关 键 词：JMJD6 水疱性口炎病毒 慢病毒包装 过表达细胞系 干扰素 

分 类 号：S852.659.6[农业科学—基础兽医学]