Tollip敲除猪肾细胞系的构建  

Construction of Tollip Knockout Pig Kidney Cell Line

作　　者：王家丽 杨帆[1,3] 邵文华 黄梦瑶 曹伟军 蒲秀瑛[2] 张伟 郑海学[1,2,3] WANG Jiali;YANG Fan;SHAO Wenhua;HUANG Mengyao;CAO Weijun;PU Xiuying;ZHANG Wei;ZHENG Haixue(State Key Laboratory for Animal Disease Control and Prevention,College of Veterinary Medicine,Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China;College of Life Science and Engineering,Lanzhou University of Technology,Lanzhou 730050,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国家口蹄疫参考实验室,兰州730000 [2]兰州理工大学生命科学与工程学院,兰州730050 [3]甘肃省病原生物学基础学科研究中心,兰州730046

出　　处：《畜牧兽医学报》2024年第4期1810-1818,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

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(21JR7RA026);兰州大学中央高校基本科研业务费专项资金(lzujbky-2022-ey20);甘肃省科技重大专项(22ZD6NA001);国家生猪产业技术体系(CARS-35)。

摘　　要：旨在探究Toll作用蛋白(toll-interacting protein, Tollip)对口蹄疫病毒(foot-and-mouth disease virus, FMDV)增殖的影响,利用CRISPR/Cas9基因编辑技术构建Tollip基因缺失的猪肾细胞系(porcine kidney cell line, PK-15),并通过PCR测序和Western blot确认敲除效果,然后利用CCK-8试剂盒测定Tollip缺失后细胞活力变化,确认敲除细胞与野生细胞无显著差异,用FMDV感染敲除细胞后,采用Western blot、间接免疫荧光、实时荧光定量和病毒滴度测定等方法测定病毒在敲除细胞系中复制情况。结果显示:感染FMDV后,敲除细胞与野生细胞相比,显著促进了病毒的复制。综上,在PK-15细胞上,成功构建Tollip缺失细胞系,且试验表明Tollip的缺失有助于FMDV的复制。研究结果为后续Tollip功能研究及抗病毒机制研究提供理论依据。In order to explore the effect of Toll-interacting protein(Tollip)on the proliferation of Foot-and-mouth disease virus(FMDV),CRISPR/Cas9 gene editing technology was used to construct a porcine kidney cell line(PK-15)with Tollip gene deletion,and the knockout effect was confirmed by PCR sequencing and Western blot,Then,CCK-8 kit was used to determine the change of cell viability after Tollip deletion,and there was no obvious difference between knockout cells and wild cells.and after knockout cells with FMDV infection,Western blot,indirect immunofluorescence,real-time fluorescence and viral titer were used to determine the replication of viruses in knockout cell lines.The results showed that after infection with FMDV,knockout cells significantly promoted virus replication compared with wild cells.Western blot,indirect immunofluorescence,real-time fluorescence and viral titer were used to determine the replication of viruses in knockout cell lines during FMDV infection.The results showed that knockout cells significantly promoted virus replication compared with wild cells during FMDV infection.In summary,Tollip deletion cell lines were successfully constructed on PK-15 cells,and experiments the results showed that Tollip deletion contributed to FMDV replication.These results provide a theoretical basis for the subsequent study of Tollip function and antiviral mechanism.

关 键 词：CRISPR/Cas9 Tollip基因 细胞系 口蹄疫病毒 

分 类 号：Q789[生物学—分子生物学]