PK-15细胞的ISG15基因敲除促进PRV的复制

doi:10.11843/j.issn.0366-6964.2022.10.033

摘要/Abstract

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ISG15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ISG15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-EP0、PRV-gE、PRV-VP16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ISG15基因；CCK-8试剂盒检测细胞活力结果表明，敲除ISG15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ISG15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ISG15可以促进PRV的复制。另外，RT-qPCR结果显示，敲除ISG15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ISG15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。

关键词: CRISPR/Cas9, ISG15, 猪伪狂犬病病毒, 基因敲除

Abstract: 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, ISG15 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-EP0, PRV-gE, PRV-VP16 and IFN-β were 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; ISG15 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 ISG15 could promote PRV replication. Besides, RT-qPCR results showed that up-regulation transcription of IFN-β induced by PRV infection was resisted in PK15-ISG15^-/- cells. In conclusion, the above results indicate that ISG15 inhibits PRV replication in PK-15 cells, which might be linked to the IFN signaling pathway.

Key words: CRISPR/Cas9, interferon stimulated gene 15, porcine pseudorabies virus, gene knockout

中图分类号:

S852.659.1

李琛, 何文峰, 赵丽娜, 凡启, 杨国庆, 刘慧敏. PK-15细胞的ISG15基因敲除促进PRV的复制[J]. 畜牧兽医学报, 2022, 53(10): 3621-3630.

LI Chen, HE Wenfeng, ZHAO Lina, FAN Qi, YANG Guoqing, LIU Huimin. Effect of Interferon Stimulated Gene 15 Knockout in PK-15 Cell Line on Replication of Pseudorabies Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3621-3630.

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