Constructing MARC-145 cell lines with <italic style="font-style: italic">NCOA4</italic> gene knockout based on CRISPR/Cas9 system and its effect on replication of HP-PRRSV

GUAN Kaifeng; YUAN Chenyang; LÜ Rongzhou; ZHANG Gaiping

doi:10.13300/j.cnki.hnlkxb.2025.05.014

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Constructing MARC-145 cell lines with NCOA4 gene knockout based on CRISPR/Cas9 system and its effect on replication of HP-PRRSV

Animal science·Veterinary medicine | 更新时间：2026-11-05

- Constructing MARC-145 cell lines with NCOA4 gene knockout based on CRISPR/Cas9 system and its effect on replication of HP-PRRSV
- Journal of Huazhong Agricultural University Vol. 44, Issue 5, Pages: 134-141(2025)
- 作者机构：
  
  1.北京大学现代农学院，北京 100871
  2.西北农林科技大学动物医学院，杨凌 712100
  3.河南省农业科学院动物免疫学重点实验室，郑州 450002
- 作者简介：
- 基金信息：
- DOI：10.13300/j.cnki.hnlkxb.2025.05.014
  CLC： S852.651
- Received：27 September 2024，
  
  Published：15 September 2025
- 稿件说明：
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官凯锋，袁晨阳，吕镕州，等.基于CRISPR/Cas9系统构建NCOA4敲除的MARC-145细胞系及其对HP-PRRSV复制的影响［J］.华中农业大学学报，2025，44（5）：134‐141.

GUAN Kaifeng,YUAN Chenyang,LÜ Rongzhou,et al.Constructing MARC-145 cell lines with NCOA4 gene knockout based on CRISPR/Cas9 system and its effect on replication of HP-PRRSV[J].Journal of Huazhong Agricultural University,2025,44(05):134-141.
官凯锋，袁晨阳，吕镕州，等.基于CRISPR/Cas9系统构建NCOA4敲除的MARC-145细胞系及其对HP-PRRSV复制的影响［J］.华中农业大学学报，2025，44（5）：134‐141. DOI： 10.13300/j.cnki.hnlkxb.2025.05.014.

GUAN Kaifeng,YUAN Chenyang,LÜ Rongzhou,et al.Constructing MARC-145 cell lines with NCOA4 gene knockout based on CRISPR/Cas9 system and its effect on replication of HP-PRRSV[J].Journal of Huazhong Agricultural University,2025,44(05):134-141. DOI： 10.13300/j.cnki.hnlkxb.2025.05.014.

摘要

为探究NCOA4介导的铁自噬在PRRSV感染中的作用，利用CRISPR/Cas9技术在PRRSV易感的MARC-145细胞中敲除铁自噬的关键受体

NCOA4

基因，并结合Sanger测序和Western blot检测

NCOA4

的敲除效率。同时运用流式细胞术、qPCR、Western blot、TCID

等对

NCOA4

缺失型和野生型MARC-145进行细胞周期、细胞活性检测，以及检测

NCOA4

缺失对PRRSV复制的影响。结果显示，本研究成功构建

NCOA4

基因敲除的MARC-145细胞系。此外，相比于野生型MARC-145细胞系，敲除

NCOA4

显著增加PRRSV

ORF7

mRNA、N蛋白及病毒滴度水平，同时促进FTH 1蛋白表达

。结果表明，铁自噬的关键受体基因

NCOA4

缺失能促进PRRSV复制。

Abstract

Ferritinophagy is a crucial cellular pathway that selectively degrades ferritin to release free iron and plays a key role in maintaining intracellular iron homeostasis. Recent studies have shown a close relationship between viral infections and host iron metabolism. As a significant pathogen severely affecting the global swine industry， the relationship between PRRSV replication and host ferritinophagy remains unclear.CRISPR/Cas9 technology was used to knockout the

NCOA4

gene encoding a key receptor for ferritinophagy in PRRSV-susceptible MARC-145 cells to study the role of

NCOA4

-mediated ferritinophagy in the infection of PRRSV. The efficiency of knockouting the

NCOA4

gene was detected with Sanger sequencing and Western blot. Methods including flow cytometry， QPCR， Western blot and TCID

assays were used to detect the cell cycle and cell viability of the

NCOA4

deficient mutant and wild-type MARC-145， and study the effect of

NCOA4

deficiency on the replication of PRRSV. The results showed that MARC-145 cell lines with

NCOA4

gene knockout were successfully constructed. The level of ORF7 mRNA， N protein， and viral titers of PRRSV in MARC-145 cell lines with

NCOA4

gene knockout was significantly increased and the expression of FTH1 protein was promoted compared with that in wild-type MARC-145 cells. It is indicated that the deletion of

NCOA4

， a key receptor gene for ferritinophagy， can promote the replication of PRRSV. It will provide a theoretical basis for the prevention and control of PRRSV.

关键词

Keywords

references

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Constructing MARC-145 cell lines with NCOA4 gene knockout based on CRISPR/Cas9 system and its effect on replication of HP-PRRSV

DOI：10.13300/j.cnki.hnlkxb.2025.05.014

摘要

Abstract

关键词

Keywords

references