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_(50)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。
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_(50) 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.
作者
官凯锋
袁晨阳
吕镕州
张改平
GUAN Kaifeng;YUAN Chenyang;LÜRongzhou;ZHANG Gaiping(School of Advanced Agricultural Sciences,Peking University,Beijing 100871,China;College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China;Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China)
出处
《华中农业大学学报》
北大核心
2025年第5期134-141,共8页
Journal of Huazhong Agricultural University
基金
国家自然科学基金项目(32302844)
中国博士后科学基金面上项目(2024M750114)。
