中国农业大学学报2025，Vol.30Issue(5)：169-178,10.DOI:10.11841/j.issn.1007-4333.2025.05.14
稳定表达IRF2基因DF-1细胞株的建立及其对H9N2 AIV复制的影响
Establishment of a DF-1 cell line stably expressing the IRF2 gene and its effect on H9N2 AIV replication
摘要
Abstract
To establish a stable DF-1 cell line expressing Interferon regulatory factor 2(IRF2),this study first successfully constructed a recombinant lentivirus targeting IRF2.Subsequently,the recombinant lentivirus was used to infect DF-1 cells,and IRF2 overexpressing polyclonal cell lines were obtained through puromycin selection and limited dilution method.The constructed cell line was subjected to viability assays,Real-time quantitative PCR(qPCR),and Western blot to verify the in vitro expression efficiency of IRF2.Viral proliferation assays were conducted to assess the impact of this cell line on H9N2 AIV replication,and finally,a dual luciferase reporter system was used to verify the regulatory effects of IRF2 on interferon-stimulated genes.The results indicated the following:1)Cell viability assays showed that stable expression of IRF2 had no significant effect on cell activity(P＞0.05).2)Real-time qPCR and Western blot demonstrated that the mRNA and protein levels of IRF2 were significantly higher in the selected cell line compared to control cells(P＜0.001).3)Real-time qPCR(P＜0.001),Western blot(P＜0.01),and TCID50(P＜0.01)in vitro assays confirmed that the overexpression of IRF2 in the DF-1-IRF2 cell line promoted the proliferation of H9N2 AIV.4)The dual luciferase reporter system results indicated that IRF2 could significantly inhibit the activities of ISRE(P＜0.01),NF-κB(P＜0.01),and IRF7(P＜0.05).In summary,this study successfully constructed a DF-1-IRF2 cell line capable of stably expressing the IRF2 gene,which is able to enhance the replication level of H9N2 AIV,and provide a basis for the subsequent study of the role of IRF2 in the replication mechanism of H9N2 AIV.关键词
H9N2 AIV/慢病毒载体/DF-1细胞/IRF2Key words
H9N2AIV/lentivirus vector/DF-1 cell/IRF2分类
农业科技引用本文复制引用
晋晨阳,马露露,郭梓轩,赵青青,代曼曼..稳定表达IRF2基因DF-1细胞株的建立及其对H9N2 AIV复制的影响[J].中国农业大学学报,2025,30(5):169-178,10.基金项目
国家自然科学基金面上项目(32473060) （32473060）
广东省自然科学基金项目(2022A1515012480,2024A1515013151) （2022A1515012480,2024A1515013151）
高等学校学科创新引智计划项目(D20008) （D20008）
特定高校学科建设专项(2023B10564003) （2023B10564003）
