Toll样受体7基因敲除对水疱性口炎病毒增殖的影响

doi:10.16431/j.cnki.1671-7236.2022.06.001

Abstract

Abstract: 【Objective】 The objective of this study was to investigate the effects of Toll-like receptor 7(TLR7)gene knockdown on the replication of Vesicular stomatitis virus (VSV).【Method】 The stable porcine renal epithelial cells (PK15) with TLR7 gene knockout were constructed using lentivirus-mediated CRISPR/Cas9 gene editing technique.The recombinant plasmid pTLR7-sgRNA was constructed and transfected into HEK293T/17 cells.Lentivirus was harvested and infected with PK15 cells.PK15-TLR7^-/- polyclonal cells were obtained after screening by purinomycin.PK15-TLR7^-/- monoclonal stable cell lines were obtained by limited dilution method after Western blotting.To verify the successful construction of TLR7 gene stabilized cell lines, optical microscopy and cytotoxicity test (CCK-8) were used to observe and detect the differences in morphology and viability of PK15 and PK15-TLR7^-/- cells.The proliferation of VSV-GFP infected PK15 and PK15-TLR7^-/- cells was observed by inverted fluorescence microscope and flow cytometry.Western blotting and Real-time quantitative PCR were used to detect the expression of GFP protein and VSV-N gene mRNA in PK15 and PK15-TLR7^-/- cells infected with VSV-GFP.Virus titers were used to detect the progenitor virus production of PK15 and PK15-TLR7^-/- cells infected with VSV-GFP.【Result】 Three sgRNAs constructed by CRISPR/Cas9 gene editing technology could effectively edit TLR7, and sgRNA2 had the highest editing efficiency.However, TLR7 knockout did not affect the morphology and viability of PK15 cells.When infected with VSV-GFP, the fluorescence intensity increased with time, and the GFP fluorescence intensity of PK15-TLR7^-/- cells was stronger than that of PK15 cells.Flow cytometry results showed that the proportion of PK15-TLR7^-/- cells infected with VSV-GFP was significantly or extremely significantly higher than that of PK15 cells at the same time (P<0.05;P<0.01).Real-time quantitative PCR results showed that the mRNA relative expression of VSV-N gene increased gradually with time from 4 to 36 h after infection, and the mRNA relative expression of VSV-N gene in PK15 cells was significantly or extremely significantly lower than that in PK15-TLR7^-/- cells (P<0.05;P<0.01).Western blotting results showed that VSV-GFP GFP was expressed in PK15 and PK15-TLR7^-/- cells at 6 h, and the expression level increased gradually with time, and the content of VSV-GFP GFP in PK15-TLR7^-/- cells was higher than that in PK15 cells.After 6 h of VSV-GFP infection, the progeny virus was released and the titer of VSV-GFP progeny virus in PK15 cells was lower than that in PK15-TLR7^-/- cells (P>0.05).The virus titer of VSV-GFP progeny in PK15 cells was significantly or extremely significantly lower than that in PK15-TLR7^-/- cells with increasing infection time (P<0.05;P<0.01).【Conclusion】 TLR7 gene knockout could promote VSV replication in PK15 cells, which preliminarily verified the role of TLR7 in natural immunity, providing new ideas and strategies for the prevention and control of VSV and other RNA viral diseases.

Key words: CRISPR/Cas9; TLR7 gene; porcine renal epithelial cells (PK15); Vesicular stomatitis virus (VSV)

CLC Number:

S852.65⁺9.5

MENG Jiejie, SONG Yue, FAN Wenjie, YANG Le, XING Jiayou, WANG Jiang, CHU Beibei, YANG Guoyu, WANG Mengdi. Effect of Toll-like Receptor 7 Gene Knockout on Proliferation of Vesicular Stomatitis Virus[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2011-2021.

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Effect of Toll-like Receptor 7 Gene Knockout on Proliferation of Vesicular Stomatitis Virus

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