PK-15细胞的ISG15基因敲除促进PRV的复制  

Effect of Interferon Stimulated Gene 15 Knockout in PK-15 Cell Line on Replication of Pseudorabies Virus

作　　者：李琛 何文峰 赵丽娜 凡启 杨国庆[1] 刘慧敏[1] LI Chen;HE Wenfeng;ZHAO Lina;FAN Qi;YANG Guoqing;LIU Huimin(College of Life Science,Henan Agricultural University,Zhengzhou 450002,China)

机构地区：[1]河南农业大学生命科学学院,郑州450002

出　　处：《畜牧兽医学报》2022年第10期3621-3630,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31902268);2021年度河南省高等学校青年骨干教师培养计划(2021GGJS034)。

摘　　要：本试验旨在研究干扰素刺激基因15(interferon-stimulated gene 15,ISG15)敲除对猪伪狂犬病病毒(PRV)复制的影响。通过CRISPR/Cas9技术构建猪ISG 15基因敲除猪肾上皮(PK-15)细胞系,利用CCK-8试剂盒检测PK-15敲除ISG 15基因对细胞活力的影响,采用间接免疫荧光技术检测PK-15以及PK15-ISG15-/-细胞感染PRV的增殖差异,通过RT-qPCR检测PRV-EP 0、PRV-gE、PRV-VP 16和IFN-β的转录水平,Western blot检测PRV-gE和ISG15的蛋白表达水平,以及通过病毒噬斑检测对子代病毒感染力的影响。结果表明,sgRNA1和sgRNA2均成功敲除ISG 15基因;CCK-8试剂盒检测细胞活力结果表明,敲除ISG 15基因对PK-15细胞活力无影响;间接免疫荧光检测结果表明,PRV感染后,PK15-ISG15-/-细胞中的荧光强度明显高于PK-15细胞;RT-qPCR和Western blot结果表明,敲除ISG 15可以促进PRV的转录和蛋白表达;病毒噬斑试验进一步显示,敲除ISG 15可以促进PRV的复制。另外,RT-qPCR结果显示,敲除ISG 15可以抑制PRV感染引起的IFN-β转录上调。本研究成功构建了PK15-ISG15-/-细胞系,并通过PRV感染试验证实ISG 15基因可以抑制PRV在PK-15细胞中的增殖,并推测这种抑制作用可能与IFN通路有关。Interferon stimulated gene 15(ISG15),a gene stimulated by interferon,plays an important role during host-viral interaction.To study the effect of ISG15 on porcine pseudorabies virus(PRV)replication,in this study,ISG 15 gene knockout cell line was constructed by CRISPR/Cas9 technology.Next,the cell viability of PK15-ISG15-/-cells was monitored by CCK-8 assay.Then,the following indexes were used to comprehensively evaluate the effect of ISG15 knockout on PRV replication:fluorescence intensity of PRV was measured by indirect immunofluorescence assay(IFA);mRNA level of PRV-EP 0,PRV-gE,PRV-VP 16 and IFN-βwere detected by RT-qPCR;protein expression levels of PRV-gE and ISG15 were evaluated by Western blot;PRV titer was detected by virus plaque assay.Results were as follows:Firstly,ISG15 gene was successfully knocked out in PK-15 cell by sgRNA1 and sgRNA2;ISG 15 gene knockout had no effect on cell viability;IFA detection showed that PRV fluorescence intensity of PK15-ISG15-/-cells was higher than that of PK-15 cells after PRV infection.Moreover,RT-qPCR and Western blot results showed that PK15-ISG15-/-cells could up-regulate PRV mRNA transcription and protein translation.Viral plaque assay further showed that knockout ISG 15 could promote PRV replication.Besides,RT-qPCR results showed that up-regulation transcription of IFN-βinduced by PRV infection was resisted in PK15-ISG15-/-cells.In conclusion,the above results indicate that ISG 15 inhibits PRV replication in PK-15 cells,which might be linked to the IFN signaling pathway.

关 键 词：CRISPR/Cas9 ISG15 猪伪狂犬病病毒 基因敲除 

分 类 号：S852.659.1[农业科学—基础兽医学]