PK-15细胞的TPL2基因敲除有利于口蹄疫病毒和塞内卡病毒复制

doi:10.11843/j.issn.0366-6964.2020.05.017

Abstract

Abstract: This study aimed to construct a TPL2 (MAP3K8/COT) knockout PK-15 cell line PK-15-TPL2^-/-, and evaluate the impact of FMDV and SVA replication before and after the gene knockout. It provides good biological material for research the mechanism of TPL2 in the process of virus infection and points out the direction of increasing the production of FMDV and SVA for vaccine production. Two single guide RNAs (sg RNA) against the TPL2 gene were screened. Sg RNA was synthesized and inserted into a lentiviral expression vector. The lentivirus was packaged and infected with PK-15 cells, and single cells that had been transfected with sg RNA were sorted by flow cytometry. The DNA mutation of TPL2 in the cell line was confirmed by sequencing, and the expression of TPL2 protein in the cell line was detected by Western blot. Replication of FMDV and SVA in PK-15-TPL2^-/- cells were evaluated by IFA, RT-qPCR, Western blot and TCID₅₀. On this basis, the activation of the interferon pathway in FMDV or SVA infected PK-15-TPL2^-/- cells was studied by measuring the mRNA expression level of interferon (IFN) and IFN stimulating gene (ISG). In TPL2 knockout PK-15 cell line, the TPL2 gene had a base insertion and deletion mutation, TPL2 protein expression was not detected in PK-15-TPL2^-/- cell line. Viral copy number was measure and compared between PK-15 and PK-15-TPL2^-/- cells after FMDV and SVA infection. The results showed that the TPL2 gene knockout significantly promoted FMDV and SVA replication. At the same time, RT-qPCR further confirmed that the mRNA expressions of IFN-α, IFN-β, IFN-γ, ISG15, ISG54 and ISG56 in PK-15-TPL2^-/- cells were significantly lower than these in PK-15 cells after FMDV and SVA infection. In summary, the TPL2 knockout PK-15 cell line was constructed successfully. The TPL2 gene has antiviral effects on FMDV and SVA. Compared with control cells, the TPL2 gene knockout is more productive to FMDV and SVA replication. Results indicated that the CRISPR/Cas9 gene-editing technology can be used as an effective tool to improve virus yield for FMDV and SVA vaccine production. It provided good biological material for studying the mechanisms of TPL2 in the process of virus infection.

Key words: TPL2 gene, PK-15 cells, gene knockout, foot-and-mouth disease virus, Seneca virus

CLC Number:

S852.659.6

YAN Minghao, HAO Junhong, ZHANG Dajun, SHEN Chaochao, XU Guowei, HOU Jing, ZHANG Keshan, ZHENG Haixue, LIU Xiangtao. TPL2 Gene Knockout in PK-15 Cells Facilitates Replication of Foot-and-mouth Disease Virus and Seneca Valley Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 1060-1073.

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TPL2 Gene Knockout in PK-15 Cells Facilitates Replication of Foot-and-mouth Disease Virus and Seneca Valley Virus

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