解剖学报 ›› 2019, Vol. 50 ›› Issue (6): 741-746.doi: 10.16098/j.issn.0529-1356.2019.06.007

• 细胞和分子生物学 • 上一篇    下一篇

猪肾上皮细胞Ⅰ型干扰素受体1敲除对伪狂犬病毒复制的影响

邵科宇 张爽 段利芳 马英先 郭玉堃 李佳佳 刘晓贺 杜永坤 褚贝贝*   

  1. 河南农业大学农业农村部动物生化与营养重点开放实验室,郑州 450002
  • 收稿日期:2019-02-10 修回日期:2019-06-06 出版日期:2019-12-06 发布日期:2019-12-06
  • 通讯作者: 褚贝贝 E-mail:wanboyi2000@163.com
  • 基金资助:
    国家自然科学基金;霍英东教育基金会高等院校青年教师基金;转基因生物新品种培育重大专项;优势特色学科建设经费

Effect of porcine kidney epithelial cells type Ⅰ interferon receptor 1 knockdown on pseudorabies virus replication

SHAO Ke-yu ZHANG Shuang DUAN Li-fang MA Ying-xian GUO Yu-kun LI Jia-jia LIU Xiao-he DU Yong-kun CHU Bei-bei*   

  1. Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs, He’nan Agricultural University, Zhengzhou 450002,China
  • Received:2019-02-10 Revised:2019-06-06 Online:2019-12-06 Published:2019-12-06
  • Contact: CHU Bei-bei E-mail:wanboyi2000@163.com

摘要:

目的 探讨Ⅰ型干扰素受体1 (IFNAR1) 对伪狂犬病病毒 (PRV) 复制的影响。 方法 以慢病毒介导的CRISPR/Cas9基因编辑技术,构建猪肾上皮细胞 (PK15) 敲除IFNAR1基因的稳定细胞系。运用细胞活力检测、荧光观察、流式细胞术检测、滴度测定、Real-time PCR等技术进行IFNAR1功能验证。 结果 随着PRV感染时间延长,敲除IFNAR1基因可以显著促进PRV-TK mRNA的转录,PRV-gE蛋白的翻译以及子代病毒的毒力。 结论 IFNAR1在抑制PRV增殖中发挥重要作用。

关键词: 猪肾上皮细胞, Ⅰ型干扰素受体1, 伪狂犬病病毒, 基因编辑, 免疫印迹法, 实时定量聚合酶链反应,

Abstract:

Objective To investigate the effect of type Ⅰ interferon receptor 1 (IFNAR1) on pseudorabies virus (PRV) replication. Methods A stable cell line in which porcine kidney epithelial cells (PK15) knocked out the IFNAR1 gene was constructed using a lentiviralmediated CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR) gene editing technique. The function of IFNAR1 was verified by cell viability assay, fluorescence observation, flow cytometry detection, titer determination, and Real-time PCR. Results With the prolongation of PRV infection, knocking out the IFNAR1 gene can significantly promote transcription of PRV-TK mRNA, translation of PRV-gE protein, and virulence of progeny virus. Conclusion IFNAR1 plays an important role in inhibiting the proliferation of PRV.

Key words: Porcine renal epithelial cell, Type Ⅰ interferon receptor 1, Pseudorabies virus, Gene editing, Western blotting, Real-time PCR, Porcine