应用CRISPR/Cas9敲除HeLa细胞DDX21基因对新城疫病毒复制的影响

doi:10.11843/j.issn.0366-6964.2019.07.012

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (7): 1433-1440.doi: 10.11843/j.issn.0366-6964.2019.07.012

应用CRISPR/Cas9敲除HeLa细胞DDX21基因对新城疫病毒复制的影响

武炜¹, 王飒², 孟春春¹, 仇旭升¹, 廖瑛¹, 谭磊¹, 宋翠萍¹, 刘炜玮¹, 孙英杰^1*, 丁铲^1*

1. 中国农业科学院上海兽医研究所, 上海 200241;
2. 福建农林大学动物科学学院, 福州 350002

收稿日期:2019-01-09 出版日期:2019-07-23 发布日期:2019-07-23
通讯作者: 丁铲,主要从事动物病毒分子生物学研究,E-mail:shoveldeen@shvri.ac.cn;孙英杰,主要从事动物病毒分子生物学研究,E-mail:sunyingjie@shvri.ac.cn
作者简介:武炜(1992-),女,内蒙古锡林郭勒盟人,硕士生,主要从事动物病毒分子生物学研究,E-mail:277310664@qq.com
基金资助:
国家自然科学基金面上项目（31872453）；国家自然科学基金重点项目（31530074）；国家重点研究开发计划（2018YFD0500100）

Role of DDX21 Gene in Newcastle Disease Virus Replication by Using CRISPR/Cas9-Mediated Gene Knockout Cells

WU Wei¹, WANG Sa², MENG Chunchun¹, QIU Xusheng¹, LIAO Ying¹, TAN Lei¹, SONG Cuiping¹, LIU Weiwei¹, SUN Yingjie^1*, DING Chan^1*

1. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China;
2. College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China

Received:2019-01-09 Online:2019-07-23 Published:2019-07-23

摘要/Abstract

摘要： 解旋酶家族是一类对所有生物都至关重要的酶，主要功能是基因解旋，DEAD/H-box家族RNA解旋酶是一类重要的解旋酶。DEAD-box RNA解旋酶DDX21被证实可能调控宿主细胞先天性免疫。新城疫病毒（NDV）是一种对养禽业危害严重的病原，NDV能够通过多种手段调控宿主先天性免疫，为了探讨DDX21如何调控NDV复制，本研究应用CRISPR/Cas9系统建立DDX21基因敲除的HeLa细胞系，并验证其对NDV复制的影响。针对DDX21基因共设计6条sgRNA，构建敲除载体并电转染细胞，通过药物筛选、亚克隆筛选后，以PCR和Western blot进行敲除效率的双重鉴定，并比较野生型和DDX21敲除细胞中NDV的复制效率。结果显示，由于DDX21对细胞生长至关重要，因此仅获得一株DDX21杂合子敲除细胞，Western blot结果显示DDX21表达量被显著抑制，NDV对在敲除细胞上的复制滴度明显低于野生型细胞，说明DDX21发挥正调控NDV复制的作用，本试验为DDX21调控抗病毒先天性免疫研究奠定了基础。

Abstract: The helicase family is a class of enzymes that are essential to all living organisms. The main function of the helicase family is gene unwapping. The DEAD/H-box family RNA helicase is a kind of important helicase. DEAD-box RNA helicase DDX21 has been proved that it may regulate innate immunity of host cells. Newcastle disease virus (NDV) is a kind of pathogen which is harmful to poultry industry. NDV can regulate the innate immunity of the host by various means. In order to verify how DDX21 regulates NDV replication, this study used CRISPR/Cas9 system to establish a DDX21 knockout HeLa cell line, and to verify its effect on NDV infection. Six sgRNAs were designed to construct knockout vectors for DDX21 gene and electrotransfected into HeLa cells. After drug screening and subcloning, the knockout efficiency was identified by PCR and Western blot, and the replication efficiency of NDV in wild type and DDX21 knockout cells was compared. The results showed that only one DDX21 heterozygote knockout cell line was obtained because DDX21 is critical for cell growth. Western blot results showed that the expression of DDX21 was significantly inhibited. The infection ability of NDV to knockout cells was significantly lower than that of wild type cells, indicating that DDX21 played a positive role in regulating NDV replication. The research above laid a foundation for the study of DDX21 regulating antiviral innate immunity.

中图分类号:

S852.659.5

武炜, 王飒, 孟春春, 仇旭升, 廖瑛, 谭磊, 宋翠萍, 刘炜玮, 孙英杰, 丁铲. 应用CRISPR/Cas9敲除HeLa细胞DDX21基因对新城疫病毒复制的影响[J]. 畜牧兽医学报, 2019, 50(7): 1433-1440.

WU Wei, WANG Sa, MENG Chunchun, QIU Xusheng, LIAO Ying, TAN Lei, SONG Cuiping, LIU Weiwei, SUN Yingjie, DING Chan. Role of DDX21 Gene in Newcastle Disease Virus Replication by Using CRISPR/Cas9-Mediated Gene Knockout Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(7): 1433-1440.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2019.07.012

https://www.xmsyxb.com/CN/Y2019/V50/I7/1433

参考文献

[1] WU Y. Unwinding and rewinding:double faces of helicase?[J]. J Nucleic Acids, 2012, 2012:140601.
[2] UMATE P, TUTEJA N, TUTEJA R. Genome-wide comprehensive analysis of human helicases[J]. Commun Integr Biol, 2011, 4(1):118-137.
[3] TANNER N K, LINDER P. DExD/H box RNA helicases:from generic motors to specific dissociation functions[J]. Mol Cell, 2001, 8(2):251-262.
[4] ABDELHALEEM M, MALTAIS L, WAIN H. The human DDX and DHX gene families of putative RNA helicases[J]. Genomics, 2003, 81(6):618-622.
[5] YONEYAMA M, KIKUCHI M, NATSUKAWA T, et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses[J]. Nat Immunol, 2004, 5(7):730-737.
[6] SAITO T, HIRAI R, LOO Y M, et al. Regulation of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2[J]. Proc Natl Acad Sci U S A, 2007, 104(2):582-587.
[7] YONEYAMA M, KIKUCHI M, MATSUMOTO K, et al. Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity[J]. J Immunol, 2005, 175(5):2851-2858.
[8] VALIENTE-ECHEVERRÍA F, HERMOSO M A, SOTO-RIFO R. RNA helicase DDX3:at the crossroad of viral replication and antiviral immunity[J]. Rev Med Virol, 2015, 25(5):286-299.
[9] ZHAO S C, GE X N, WANG X L, et al. The DEAD-box RNA helicase 5 positively regulates the replication of porcine reproductive and respiratory syndrome virus by interacting with viral Nsp9in vitro[J]. Virus Res, 2015, 195:217-224.
[10] DONG Y C, YE W, YANG J, et al. DDX21 translocates from nucleus to cytoplasm and stimulates the innate immune response due to dengue virus infection[J]. Biochem Biophys Res Commun, 2016, 473(2):648-653.
[11] FLORES-ROZAS H, HURWITZ J. Characterization of a new RNA helicase from nuclear extracts of HeLa cells which translocates in the 5' to 3' direction[J]. J Biol Chem, 1993, 268(28):21372-21383.
[12] ZHANG Z Q, KIM T, BAO M S, et al. DDX1, DDX21, and DHX36 helicases form a complex with the adaptor molecule TRIF to sense dsRNA in dendritic cells[J]. Immunity, 2011, 34(6):866-878.
[13] CHEN G F, LIU C H, ZHOU L G, et al. Cellular DDX21 RNA helicase inhibits influenza A virus replication but is counteracted by the viral NS1 protein[J]. Cell Host Microbe, 2014, 15(4):484-493.
[14] ALEXANDER D J. Newcastle disease and other avian paramyxoviruses[J]. Rev Sci Tech, 2000, 19(2):443-462.
[15] FIOLA C, PEETERS B, FOURNIER P, et al. Tumor selective replication of Newcastle disease virus:association with defects of tumor cells in antiviral defence[J]. Int J Cancer, 2006, 119(2):328-338.
[16] HSU P D, LANDER E S, ZHANG F. Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 157(6):1262-1278.
[17] YU Z S, REN M D, WANG Z X, et al. Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila[J]. Genetics, 2013, 195(1):289-291.
[18] HODROJ D, SERHAL K, MAIORANO D. Ddx19 links mRNA nuclear export with progression of transcription and replication and suppresses genomic instability upon DNA damage in proliferating cells[J]. Nucleus, 2017, 8(5):489-495.
[19] SONG C L, HOTZ-WAGENBLATT A, VOIT R, et al. SIRT7 and the DEAD-box helicase DDX21 cooperate to resolve genomic R loops and safeguard genome stability[J]. Genes Dev, 2017, 31(13):1370-1381.
[20] CALO E, FLYNN R A, MARTIN L, et al. RNA helicase DDX21 coordinates transcription and ribosomal RNA processing[J]. Nature, 2015, 518(7538):249-253.
[21] MA Z, MOORE R, XU X X, et al. DDX24 negatively regulates cytosolic RNA-mediated innate immune signaling[J]. PLoS Pathog, 2013, 9(10):e1003721.
[22] JANKNECHT R. Multi-talented DEAD-box proteins and potential tumor promoters:p68 RNA helicase (DDX5) and its paralog, p72 RNA helicase (DDX17)[J]. Am J Transl Res, 2010, 2(3):223-234.
[23] QIU X S, FU Q, MENG C C, et al. Newcastle disease virus V protein targets phosphorylated STAT1 to block IFN-I signaling[J]. PLoS One, 2016, 11(2):e0148560.
[24] SUN Y J, DONG L N, YU S Q, et al. Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation[J]. FASEB J, 2017, 31(4):1337-1353.
[25] SUN Y J, YU S Q, DING N, et al. Autophagy benefits the replication of Newcastle disease virus in chicken cells and tissues[J]. J Virol, 2014, 88(1):525-537.

应用CRISPR/Cas9敲除HeLa细胞DDX21基因对新城疫病毒复制的影响

Role of DDX21 Gene in Newcastle Disease Virus Replication by Using CRISPR/Cas9-Mediated Gene Knockout Cells

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	孟令宅, 陈春丽, 于蒙蒙, 王占新, 王素艳, 刘鹏, 何塔娜, 郭茹, 陈运通, 刘长军, 祁小乐, 吴志强, 高玉龙. B亚型禽偏肺病毒对黄羽肉鸡致病性分析及其灭活疫苗免疫效果评价[J]. 畜牧兽医学报, 2023, 54(12): 5154-5161.
[2]	蒋盛强, 胡靖, 陈红英. H1N1亚型流感病毒感染A549细胞的环状RNA表达分析[J]. 畜牧兽医学报, 2023, 54(11): 4724-4734.
[3]	张傲, 谭斌, 刘可欣, 刘佳利, 张淑琴. 一株H1N1亚型猪流感病毒全基因组特征分析[J]. 畜牧兽医学报, 2023, 54(11): 4866-4871.
[4]	何辰香, 高闪电, 田占成, 独军政, 王锦明, 关贵全, 殷宏. 基于G_NS蛋白的牛流行热病毒感染与免疫鉴别诊断ELISA方法的建立[J]. 畜牧兽医学报, 2023, 54(10): 4320-4326.
[5]	常益铭, 汤承, 岳华. 牦牛源牛呼吸道合胞体病毒的分子流行病学调查[J]. 畜牧兽医学报, 2023, 54(5): 2030-2041.
[6]	方源, 侯巧弟, 项超辉, 赵红奕, 齐雪峰. IFITM3对小反刍兽疫病毒在山羊子宫内膜上皮细胞中增殖的调控效应[J]. 畜牧兽医学报, 2023, 54(5): 2200-2207.
[7]	李舜, 李桂珍, 原耀贤, 李康健, 马春全, 李守军, 黄良宗, 马骏. PA-X基因的长度变化对H3N2犬流感病毒复制能力和致病性的影响[J]. 畜牧兽医学报, 2023, 54(1): 272-280.
[8]	崔明仙, 王星博, 黄彦铭, 卞希一, 冯梦珂, 颜焰, 董伟仁, 周继勇. 3株H3N2亚型禽流感病毒的基因组特征与演化分析[J]. 畜牧兽医学报, 2022, 53(11): 4116-4122.
[9]	于蒙蒙, 包媛玲, 王素艳, 辛子琪, 刘鹏, 冯笑艳, 孟令宅, 郭茹, 张艳萍, 刘长军, 祁小乐, 李俊平, 王笑梅, 高玉龙. B亚型禽偏肺病毒的分离鉴定及致病性研究[J]. 畜牧兽医学报, 2022, 53(10): 3540-3549.
[10]	周勇, 李知新, 鲁宏伟, 孙燕, 李甜, 杜凡姝, 蒲娟. 我国H5和H7N9亚型高致病性禽流感的监测及疫情暴发分析[J]. 畜牧兽医学报, 2022, 53(9): 3093-3106.
[11]	潘春容, 邓瑞雪, 胡林杰, 朱学亮, 孙跃峰, 王桂荣, 蒙学莲. 组蛋白去乙酰化酶抑制剂对小反刍兽疫病毒复制的影响[J]. 畜牧兽医学报, 2022, 53(7): 2307-2316.
[12]	陈子轩, 张楠, 胡群, 全柯吉, 秦涛, 陈素娟, 彭大新, 刘秀梵. 我国H9N2亚型禽流感病毒血凝素蛋白145和153位抗原位点变异分析[J]. 畜牧兽医学报, 2022, 53(4): 1165-1172.
[13]	段志强, 谢玲玲, 陈佳琪, 唐宏, 王燕碧, 赵采芹, 赵佳福. 新城疫病毒M蛋白与宿主蛋白相互作用的研究进展[J]. 畜牧兽医学报, 2022, 53(1): 32-42.
[14]	严雅瑶, 顾敏, 刘秀梵. HA蛋白位点变异影响H7N9亚型流感病毒特性的研究进展[J]. 畜牧兽医学报, 2021, 52(8): 2093-2106.
[15]	蒋梅, 陈武, 翟俊琼, 卜婉迪, 谢逸伦, 刘灿彬, 单芬, 罗满林. 小熊猫源犬瘟热病毒株H、F基因的克隆及序列分析[J]. 畜牧兽医学报, 2021, 52(6): 1670-1676.