| Vesicular stomatitis(VS)is an acute zoonotic infectious disease,and the genome of vesicular stomatitis virus(VSV)is highly mutated RNA,which makes the prevention and control of the disease difficult.In order to resist the invasion of a variety of pathogenic microorganisms,the body evolved an immune system,and innate immunity is the first barrier to protect the health of the body.Interferon has broad-spectrum antiviral effect,and plasmacytoid dendritic cells(p DC)are the professional cells producing type I interferon.In addition,proinflammatory cytokines also play an important role in the inflammatory response.The endosomal Toll-like receptor 7(TLR7)recognizes the single stranded RNA ligand of the virus and activates the p DC to produce a large amount of type I interferon and a variety of pro-inflammatory cytokines through signaling cascades.In this experiment,TLR7 gene was knocked out to study the effects on the proliferation of VSV.Then VSV was used as the experimental material and TLR7 knockout cell line was used as the tool to study the antiviral effect and target of TLR716.The results showed that TLR716 had better antiviral effect than TLR701,and TLR716 was preliminarily proved to be an agonist of TLR7.First,the toxicity of TLR701 or TLR716 to PK15 and HEK293T/17 cells was detected,and no toxicity was found in the concentration range of 0-2.0 μM.Then TLR701 and TLR716 were tested for half of the effective inhibition concentration of VSV,and the selectivity coefficients of the two were calculated.The preliminary screening of their antiviral effect showed that TLR716 had better antiviral effect.The effects of TLR701 and TLR716 on the proliferation of VSV in PK15 cells were detected by flow cytometry.The results showed that TLR701 and TLR716 promoted the proliferation of VSV in PK15 cells,and the effect of TLR716 was better at the same concentration.In this study,lentivirus-mediated CRISPR/Cas9 gene editing technique was used to construct PK15-TLR7-/-cell lines.First,the porcine TLR7 gene sequence was found and designed using sg RNA online design software.After being connected with lenti CRISPRv2 vector,the sg RNA was packaged with lentivirus.After puromycin screening,the gene editing efficiency was detected by Western blot,and the polyclonal cell line with the highest editing efficiency was selected.After limited dilution,monoclonal cell lines were obtained.Western blot was used to detect the expression of TLR7 protein,and the genome was extracted and sent to the company for sequencing.The effect of TLR7 knockout on cell morphology was observed by microscopy,and the effect of TLR7 knockout on cell viability was detected using CCK-8 kit.The results showed that PK15-TLR7-/-monoclonal cell lines were successfully obtained,and TLR7 knockout did not affect the morphology and viability of PK15 cells.Western blot,Q-PCR,flow cytometry and TCID50 were used to detect VSV replication in PK15 and PK15-TLR7-/-cells at different time points to test whether TLR7 gene knockout promoted VSV proliferation.The results showed that after knockout TLR7 gene could promote the proliferation of VSV in PK15 cell.Then flow cytometry was used to detect the effects of TLR701 or TLR716 on the proliferation of VSV in PK15-TLR7-/-cells.The results showed that neither of them played a role in PK15-TLR7-/-cells.luciferase reporting assay was used to detect the activation of TLR701 and TLR716 on the promoter of NF-κB target gene downstream of TLR7.The results showed that TLR716 could activate the promoter of NF-κB target gene downstream of TLR7.The m RNA expression levels of IL-6,TNF-α and IFN-β in PK15 and PK15-TLR7-/-cells were detected by Q-PCR.The results showed that TLR716 could up-regulate these cytokines at the transcriptional level in PK15 cells,but could not play a role in PK15-TLR7-/-cells.In summary,TLR716 is preliminarily proved to be an agonist of TLR7,and its inhibitory effect on the proliferation of VSV is superior to TLR701. |
PDF Full Text Request