Tollip敲除猪肾细胞系的构建

doi:10.11843/j.issn.0366-6964.2024.04.042

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (4): 1810-1818.doi: 10.11843/j.issn.0366-6964.2024.04.042

• 研究简报 • 上一篇

Tollip敲除猪肾细胞系的构建

王家丽^1,2, 杨帆^1,3, 邵文华¹, 黄梦瑶^1,3, 曹伟军^1,3, 蒲秀瑛², 张伟^1,3*, 郑海学^1,2,3*

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

收稿日期:2023-07-31 出版日期:2024-04-23 发布日期:2024-04-26
通讯作者: 张伟,主要从事动物病毒病致病机制研究,E-mail:zw20082008@126.com;郑海学,主要从事动物传染病学与流行病学研究,E-mail:zhenghaixue@caas.cn
作者简介:王家丽(1998-)，女，甘肃靖远人，硕士，主要从事动物病毒感染与免疫机制研究，E-mail：1816490058@qq.com
基金资助:
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)

Construction of Tollip Knockout Pig Kidney Cell Line

WANG Jiali^1,2, YANG Fan^1,3, SHAO Wenhua¹, HUANG Mengyao^1,3, CAO Weijun^1,3, PU Xiuying², ZHANG Wei^1,3*, ZHENG Haixue^1,2,3*

1. 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;
2. College of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China;
3. Gansu Province Research Center for Basic Disciplines of Pathogen Biology, Lanzhou 730046, China

Received:2023-07-31 Online:2024-04-23 Published:2024-04-26

摘要/Abstract

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。

关键词: CRISPR/Cas9, Tollip基因, 细胞系, 口蹄疫病毒

Abstract: 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. 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 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.

Key words: CRISPR/Cas9, Tollip gene, cell lines, foot-and-mouth disease virus

中图分类号:

Q789

王家丽, 杨帆, 邵文华, 黄梦瑶, 曹伟军, 蒲秀瑛, 张伟, 郑海学. Tollip敲除猪肾细胞系的构建[J]. 畜牧兽医学报, 2024, 55(4): 1810-1818.

WANG Jiali, YANG Fan, SHAO Wenhua, HUANG Mengyao, CAO Weijun, PU Xiuying, ZHANG Wei, ZHENG Haixue. Construction of Tollip Knockout Pig Kidney Cell Line[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1810-1818.

参考文献

[1] CAPELLUTO D G S. Tollip:a multitasking protein in innate immunity and protein trafficking[J]. Microbes Infect, 2012, 14(2):140-147.
[2] BURNS K, CLATWORTHY J, MARTIN L, et al. Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor[J]. Nat Cell Biol, 2000, 2(6):346-351.
[3] BAKER B, GENG, CHEN K Q, et al. Alteration of lysosome fusion and low-grade inflammation mediated by super-low-dose endotoxin[J]. J Biol Chem, 2015, 290(10):6670-6678.
[4] DIAO N, ZHANG Y, CHEN K Q, et al. Deficiency in Toll-interacting protein (Tollip) skews inflamed yet incompetent innate leukocytes in vivo during DSS-induced septic colitis[J]. Sci Rep, 2016, 6:34672.
[5] JIN S H, HE X, MA L, et al. Suppression of ACE2 SUMOylation protects against SARS-CoV-2 infection through TOLLIP-mediated selective autophagy[J]. Nat Commun, 2022, 13:5204.
[6] ZHANG D J, WANG G C, SHI X J, et al. Inactivated ORF virus inhibiting foot-and-mouth disease virus replication in BHK-21 cells[J]. Chinese Veterinary Science, 2022, 52(4):403-409. (in Chinese)
张大俊, 王国春, 史喜绢, 等. 灭活的羊口疮病毒抑制口蹄疫病毒在BHK-21细胞中的复制[J]. 中国兽医科学, 2022, 52(4):403-409.
[7] YOU W N. Generation of double gene specific knock-in pigs at Rosa26 locus[D]. Changchun:Jilin University, 2021. 尤文妮. Rosa26位点双性状基因定点整合基因编辑猪的制备[D]. 长春:吉林大学, 2021.
[8] LIANG J P, CHEN J H, SHANG R F, et al. effect of light and oxygen on the activity of hypericin protein complex against foot mouth disease virus[J]. China Animal Health, 2005(11):29-30. (in Chinese)
梁剑平, 陈积红, 尚若峰, 等. 光与氧对金丝桃素蛋白复合物抗口蹄疫病毒的活性的影响[J]. 中国动物保健, 2005(11):29-30.
[9] LI X, ZHANG F D, ZHANG Z W, et al. Recent advance on foot-and-mouth disease virus utilizes self-proteins to evade innate immunity response of host[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(11):2622-2632. (in Chinese)
李显, 张富东, 张中旺, 等. 口蹄疫病毒利用自身蛋白逃逸宿主天然免疫应答的研究进展[J]. 畜牧兽医学报, 2020, 51(11):2622-2632.
[10] LIU K. Establishment and in vitro experimental study of CRISPR/Cas9-mediated transcriptional activation system[D]. Xi'an:Shaanxi Normal University, 2016. (in Chinese)
刘宽. CRISPR/Cas9介导的转录激活系统的建立及其体外实验研究[D]. 西安:陕西师范大学, 2016.
[11] ZHENG L Y. Optimization of crop breeding using CRISPR/Cas9 gene editing technology[J]. Fujian Science & Technology of Tropical Crops, 2019, 44(2):40-44. (in Chinese)
郑丽瑶. 利用CRISPR/Cas9基因编辑技术优化作物育种[J]. 福建热作科技, 2019, 44(2):40-44.
[12] CHEN C Y, LIU A W, ZHU X Y, et al. Research progress on the relationship between hepatitis B virus genome variation and occult infection[J]. The Medical Forum, 2006, 10(1):85-86. (in Chinese)
陈常云, 刘爱武, 朱新宇, 等. 乙型肝炎病毒基因组变异与隐匿性感染的关系研究进展[J]. 基层医学论坛, 2006, 10(1):85-86.
[13] YANG J T, ZHANG L Q, HE F C. The functions of Toll-like receptors and their roles in liver diseases[J]. Chinese Journal of Hepatology, 2006, 14(8):632-634. (in Chinese)
杨俊涛, 张令强, 贺福初. Toll样受体在肝脏疾病中的功能[J]. 中华肝脏病杂志, 2006, 14(8):632-634.
[14] KAWAI T, AKIRA S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity[J]. Immunity, 2011, 34(5):637-650.
[15] MA Z C, BAI J, JIANG C L, et al. Tegument protein UL21 of alpha-herpesvirus inhibits the innate immunity by triggering CGAS degradation through TOLLIP-mediated selective autophagy[J]. Autophagy, 2023, 19(5):1512-1532.
[16] XU Z, JIN Y, GAO Z Z, et al. Autophagic degradation of CCN2(cellular communication network factor 2) causes cardiotoxicity of sunitinib[J]. Autophagy, 2022, 18(5):1152-1173.
[17] DAKHAMA A, AL MUBARAK R, PAVELKA N, et al. Tollip inhibits ST2 signaling in airway epithelial cells exposed to type 2 cytokines and rhinovirus[J]. J Innate Immun, 2020, 12(1):103-115.
[18] LIU X M. Tollip orchestrates macrophage polarization to alleviate intestinal mucosal inflammation[D]. Guangzhou:Southern Medical University, 2022. (in Chinese)
刘晓明. Tollip通过调控巨噬细胞极化减轻肠道黏膜炎症研究[D]. 广州:南方医科大学, 2022.
[19] YU Y M. Effects of puerarin on growth performance and intestinal barrier function of broilers fed oxidized soybean oil[D]. Nanchang:Jiangxi Agricultural University, 2021. (in Chinese)
余应梅. 葛根素对饲喂氧化大豆油肉鸡生长性能和肠道屏障功能的影响[D]. 南昌:江西农业大学, 2021.
[20] WANG T, LUO R, ZHANG J, et al. The MGF300-2R protein of African swine fever virus is associated with viral pathogenicity by promoting the autophagic degradation of IKKα and IKKβ through the recruitment of TOLLIP[J]. PLoS Pathog, 2023, 19(8):e1011580.
[21] YANG J, LI M, ZHENG Q C. Emerging role of Toll-like receptor 4 in hepatocellular carcinoma[J]. J Hepatocell Carcinoma, 2015, 2:11-17.

Tollip敲除猪肾细胞系的构建

Construction of Tollip Knockout Pig Kidney Cell Line

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