干扰素调节因子敲减细胞系的构建及其对猪伪狂犬病病毒增殖的影响

doi:10.11843/j.issn.0366-6964.2024.09.034

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (9): 4100-4109.doi: 10.11843/j.issn.0366-6964.2024.09.034

干扰素调节因子敲减细胞系的构建及其对猪伪狂犬病病毒增殖的影响

付艺乾¹^,²^,³(), 梁东阁¹^,²^,³, 王铭洋¹^,²^,³, 潘佳佳¹^,²^,³, 杨彦宾¹^,²^,³, 曾磊¹^,²^,³^,*(), 康相涛⁴^,*()

1. 河南农业大学动物医学院，郑州 450046
2. 河南农业大学，农业农村部动物生化与营养重点实验室，郑州 450046
3. 河南农业大学，河南省动物生长发育调控重点实验室，郑州 450046
4. 河南农业大学动物科技学院，郑州 450046

收稿日期:2023-11-14 出版日期:2024-09-23 发布日期:2024-09-27
通讯作者: 曾磊,康相涛 E-mail:1050223216@qq.com;zenglei2021918@163.com;xtkang2001@263.net
作者简介:付艺乾(2000-)，男，河南驻马店人，硕士，主要从事动物生物化学与分子生物学研究，E-mail: 1050223216@qq.com
基金资助:
河南农业大学博士启动基金资助项目(30501221);河南省高等教育教学改革研究与实践项目(2021SJGLX351)

Construction of Interferon Regulatory Factor Knockdown Cell Line and Its Effect on Pseudorabies Virus Proliferation

Yiqian FU¹^,²^,³(), Dongge LIANG¹^,²^,³, Mingyang WANG¹^,²^,³, Jiajia PAN¹^,²^,³, Yanbin YANG¹^,²^,³, Lei ZENG¹^,²^,³^,*(), Xiangtao KANG⁴^,*()

1. College of Animal Medicine, Henan Agricultural University, Zhengzhou 450046, China
2. Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Areas, Henan Agricultural University, Zhengzhou 450046, China
3. Henan Key Laboratory of Animal Growth and Development Regulation, Henan Agricultural University, Zhengzhou 450046, China
4. College of Animal Science and Technology, Henan Agricultural University, Zhengzhou 450046, China

Received:2023-11-14 Online:2024-09-23 Published:2024-09-27
Contact: Lei ZENG, Xiangtao KANG E-mail:1050223216@qq.com;zenglei2021918@163.com;xtkang2001@263.net

摘要/Abstract

摘要：

旨在揭示干扰素调节因子(interferon regulatory factor，IRFs)对猪伪狂犬病病毒(pseudorabies virus，PRV)增殖的影响，本研究通过shRNA技术构建敲减IRF1-9的PK15细胞系，并检测其敲减效率。采用流式细胞术以及滴度测定检测敲减IRF1-9后PRV的增殖情况；利用RT-qPCR技术检测PRV感染细胞后PRV-gB、IFN-β、ISG-15和IL-6的mRNA表达水平；Western blot技术检测PRV感染细胞后gB蛋白的表达水平。敲减效率测定结果显示，PK15细胞中的IRF1-9 mRNA表达水平均有显著降低；流式细胞术及滴度测定结果表明，敲减IRF1-9基因后有助于PRV的增殖；RT-qPCR及Western blot结果证明，敲减IRF1-9基因后，PRV-gB mRNA及蛋白表达水平均有显著提高；细胞炎性因子的mRNA表达水平检测结果发现，敲减IRF1-9抑制PRV诱导的IFN-β、ISG-15以及IL-6 mRNA表达。综上所述，IRF1-9是PRV在PK-15细胞内复制的宿主限制因子。

关键词: shRNA, 干扰素调节因子, 猪伪狂犬病病毒, 细胞炎性因子

Abstract:

To elucidate the impact of interferon regulatory factors (IRFs) on the proliferation of porcine pseudorabies virus (PRV), this study utilized shRNA technology to establish a PK15 cell line with the targeted knockdown of IRF1-9 genes. The knockdown efficiency was verified, and the proliferation of PRV subsequent to the knockdown was assessed using flow cytometry and titration assays. Additionally, the mRNA expression levels of PRV-gB, IFN-β, ISG-15, and IL-6 in PRV-infected cells were quantified by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The expression of the gB protein in PRV-infected cells was analyzed by Western blotting. The knockdown efficiency assay demonstrated a significant reduction in the expression of IRF1-9 mRNA in PK15 cells. The results from flow cytometry and titration assays indicated that the knockdown of IRF1-9 genes facilitated the proliferation of PRV. Furthermore, RT-qPCR and Western blot results revealed a significant increase in the mRNA and protein expression levels of PRV-gB following the knockdown of IRF1-9 genes. The analysis of mRNA expression levels of cellular inflammatory factors showed that the knockdown of IRF1-9 inhibited the PRV-induced expression of IFN-β, ISG-15, and IL-6 at the mRNA level. In conclusion, these findings suggest that IRF1-9 serves as a host restriction factor, limiting the replication of PRV within PK-15 cells.

Key words: shRNA, interferon regulatory factor, pseudorabies virus, inflammatory cytokines

中图分类号:

付艺乾, 梁东阁, 王铭洋, 潘佳佳, 杨彦宾, 曾磊, 康相涛. 干扰素调节因子敲减细胞系的构建及其对猪伪狂犬病病毒增殖的影响[J]. 畜牧兽医学报, 2024, 55(9): 4100-4109.

Yiqian FU, Dongge LIANG, Mingyang WANG, Jiajia PAN, Yanbin YANG, Lei ZENG, Xiangtao KANG. Construction of Interferon Regulatory Factor Knockdown Cell Line and Its Effect on Pseudorabies Virus Proliferation[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4100-4109.

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名称 Name	引物序列(5′→3′) Sequence
shIRF-1	CCGGGCTACACAGCTCAGGGATACACTCGAGTGTATCCCTGAGCTGTGTAGCTTTTTG AATTCAAAAAGCTACACAGCTCAGGGATACACTCGAGTGTATCCCTGAGCTGTGTAGC
shIRF-2	CCGGGCATCCATTCACACCGAATGCCTCGAGGCATTCGGTGTGAATGGATGCTTTTTG AATTCAAAAAGCATCCATTCACACCGAATGCCTCGAGGCATTCGGTGTGAATGGATGC
shIRF-3	CCGGGGAAAGAAGCATTGCGTTTAGCTCGAGCTAAACGCAATGCTTCTTTCCTTTTTG AATTCAAAAAGGAAAGAAGCATTGCGTTTAGCTCGAGCTAAACGCAATGCTTCTTTCC
shIRF-4	CCGGGCTTTATGCCAAGAGACTTTGCTCGAGCAAAGTCTCTTGGCATAAAGCTTTTTG AATTCAAAAAGCTTTATGCCAAGAGACTTTGCTCGAGCAAAGTCTCTTGGCATAAAGC
shIRF-5	CCGGGGACTTTCGCCTCATCTATGACTCGAGTCATAGATGAGGCGAAAGTCCTTTTTG AATTCAAAAAGGACTTTCGCCTCATCTATGACTCGAGTCATAGATGAGGCGAAAGTCC
shIRF-8	CCGGGGCTACGCTGTGCTTTGAACACTCGAGTGTTCAAAGCACAGCGTAGCCTTTTTG AATTCAAAAAGGCTACGCTGTGCTTTGAACACTCGAGTGTTCAAAGCACAGCGTAGCC

名称 Name	引物序列(5′→3′) Sequence
Q-β-actin-F	CTGAACCCCAAAGCCAACCGT
Q-β-actin-R	TTCTCCTTGATGTCCCGCACG
Q-gB-F	GGCATCGCCAACTTCTTCC
Q-gB-R	CCTCGTCCACGTCGTCCTC
Q-IFN-β-F	ATGATGGTGCCCTACACGCT
Q-IFN-β-R	TCCACCTGCTCCGTCAGCCT
Q-ISG-15-F	GGTGAGGAACGACAAGGGTC
Q-ISG-15-R	GGCCTTGAGGTCATACTCCCC
Q-IL-6-F	GCCTGAGGGCCATTCGGATA
Q-IL-6-R	TGTGCCCAGTGGACAGGTTT
Q-IRF1-F	ATCCTCTGCCTTCTCTTCGCT
Q-IRF1-R	AGCCCTGGGATTTGGTTGGA
Q-IRF2-F	AGGAAAGCATCAACCGGGAG
Q-IRF2-R	TCCGGTATACCCTGAAGGCA
Q-IRF3-F	AACCGGAAAGAAGCATTGCG
Q-IRF3-R	GGTGTCTGGCTCAGGAAAGT
Q-IRF4-F	TCCCAGCTCAGGTTCACAAC
Q-IRF4-R	TGTCCCCTGGCAACCATTTTT
Q-IRF5-F	GGCAGATGGATGGTCAAGACA
Q-IRF5-R	ACTGGCATTCAGACCACCCT
Q-IRF6-F	CCTCACCCCCGAAGAGTTC
Q-IRF6-R	GAAGCGCTTGGAGTCTCTGT
Q-IRF7-F	TCTACCCCCATCTGTGACCC
Q-IRF7-R	AGGTGTTAACCGCAGGTGAG
Q-IRF8-F	CAGCGGCTCAGGATGTGC
Q-IRF8-R	CCGGAACATGCTCTTCTGGT
Q-IRF9-F	ATCCTCCAGGACCCCTTCAA
Q-IRF9-R	AACCCTACCTTCGGAGACT

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干扰素调节因子敲减细胞系的构建及其对猪伪狂犬病病毒增殖的影响

Construction of Interferon Regulatory Factor Knockdown Cell Line and Its Effect on Pseudorabies Virus Proliferation

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