凋亡相关斑点样蛋白敲除PK-15细胞系的建立及对PRV感染的影响

doi:10.11843/j.issn.0366-6964.2021.02.020

Abstract

Abstract: This study aimed to investigate the effect of ASC gene knockout on the infection of pseudorabies virus (PRV) in PK-15 cells. The CRISPR/Cas9 gene-editing technology mediated by lentivirus was applied to knock out the ASC gene of porcine renal epithelial cell (PK-15), and the efficiency was detected with T7 endonuclease. Flow cytometry was applied to detect the difference of PRV-GFP proliferation between in PK-15 and PK15-ASC^-/- cells. The transcription levels of PRV-gB, IL-1β, IFN-β, ISG15 genes and the expression of PRV-gE protein were tested with RT-PCR and Western blot, the titer of progeny PRV was determined with TCID₅₀ method. The results showed that specific sgRNA could carry out efficient gene editing for ASC gene, and the activity of PK15-ASC^-/- cells was not significantly affected. After infection, the proliferation of PRV-GFP in PK15-ASC^-/- cells was significantly inhibited compared with that in PK-15 cells. Besides, both the transcription level of PRV-gB gene and the expression level of PRV-gE protein in PK15-ASC^-/- cells were also lower than that in PK-15 cells. Nevertheless, the transcription level of IFN-β and ISG15 gene presented the opposite results. The virus titer test showed that knockout of ASC gene could significantly inhibit the proliferation of PRV progeny virus. In this study, a PK15-ASC^-/- cell line was successfully constructed, and in which the PRV infection was significantly inhibited.

Key words: pseudorabies virus, apoptosis-associated speck-like protein containing a CARD, innate immunity, porcine kidney epithelial cells

CLC Number:

S852.659.1

ZHAI Yunyun, LI Jiajia, ZHANG Shuang, REN Ziyu, JIN Qianyue, DU Yongkun, WAN Bo. Establishment of Apoptosis Associated Dot Like Protein Knockout PK-15 Cell Line and Its Effect on Porcine Pseudorabies Virus Infection[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(2): 478-487.

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Establishment of Apoptosis Associated Dot Like Protein Knockout PK-15 Cell Line and Its Effect on Porcine Pseudorabies Virus Infection

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