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

Abstract

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

CLC Number:

S852.659.1

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

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