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IRF 1-9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IRF 1-9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IRF 1-9 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IRF 1-9基因后有助于PRV的增殖;RT-qPCR及Western blot结果证明,敲减IRF 1-9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IRF 1-9抑制PRV诱导的IFN-β、ISG-15以及IL-6 mRNA表达。综上所述,IRF 1-9是PRV在PK-15细胞内复制的宿主限制因子。
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.
作者
付艺乾
梁东阁
王铭洋
潘佳佳
杨彦宾
曾磊
康相涛
FU Yiqian;LIANG Dongge;WANG Mingyang;PAN Jiajia;YANG Yanbin;ZENG Lei;KANG Xiangtao(College of Animal Medicine,Henan Agricultural University,Zhengzhou 450046,China;Key Laboratory of Animal Biochemistry and Nutrition,Ministry of Agriculture and Rural Areas,Henan Agricultural University,Zhengzhou 450046,China;Henan Key Laboratory of Animal Growth and Development Regulation,Henan Agricultural University,Zhengzhou 450046,China;College of Animal Science and Technology,Henan Agricultural University,Zhengzhou 450046,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2024年第9期4100-4109,共10页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
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)。
