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

摘要/Abstract

摘要： 旨在构建TPL2（MAP3K8/COT）基因敲除PK-15细胞系PK-15-TPL2^-/-，评估该基因敲除前后对口蹄疫病毒（FMDV）和塞内卡病毒（SVA）复制的影响及产生影响的原因，为研究TPL2在病毒感染过程中的作用机制提供良好的生物材料，也为疫苗生产过程中进一步提升FMDV和SVA产量指明方向。筛选2条针对TPL2基因的单向导RNA（sg RNA），合成sg RNA并将其插入到含有GFP标签的慢病毒表达载体，构建sg RNA/Cas9慢病毒表达质粒，包装慢病毒并感染PK-15细胞，通过流式细胞仪分选出已被转入sg RNA的单细胞。通过测序确认细胞系中TPL2的DNA序列，通过蛋白质印迹（Western blot）方法检测细胞系中TPL2表达情况。使用FMDV和SVA感染构建好的细胞系，利用IFA、RT-qPCR、Western blot和TCID₅₀评估FMDV和SVA在PK-15-TPL2^-/-细胞中的复制水平，在此基础上通过测定干扰素（IFN）和IFN刺激基因（ISG）的mRNA表达水平，研究了FMDV或SVA感染的PK-15-TPL2^-/-细胞中干扰素途径的激活状态。TPL2基因敲除PK-15细胞系中TPL2基因发生了碱基插入突变和碱基缺失突变，构建的细胞系中均未检测到TPL2蛋白质表达。测定并比较了FMDV和SVA感染的PK-15和PK-15-TPL2^-/-细胞中病毒含量，表明TPL2基因敲除显著促进了FMDV和SVA的复制。同时，RT-qPCR进一步表明与FMDV和SVA感染期间的PK-15细胞相比，PK-15-TPL2^-/-细胞中IFN-α、IFN-β、IFN-γ、ISG15、ISG54和ISG56的mRNA表达明显降低。综上所述，本研究成功构建了TPL2基因敲除的PK-15细胞系，与对照细胞相比，TPL2基因的敲除更利于FMDV和SVA的复制，这可能与IFN-α、IFN-β、IFN-γ、ISG15、ISG54和ISG56表达的抑制有关。本研究提示CRISPR/Cas9基因编辑技术可以作为在动物和疫苗开发过程中编辑细胞系以提高病毒产量的有效工具，本结果为进一步提升FMDV和SVA产量指明了方向，也为研究TPL2在病毒感染过程中的作用机制提供了良好的生物材料。

关键词: TPL2基因, PK-15细胞, 基因敲除, 口蹄疫病毒, 塞内卡病毒

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

中图分类号:

S852.659.6

闫鸣昊, 郝军红, 张大俊, 申超超, 徐国伟, 侯景, 张克山, 郑海学, 刘湘涛. 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[J]. 畜牧兽医学报, 2020, 51(5): 1060-1073.

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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