Table 1_Seneca valley virus VP4 protein regulates the transcription of different cytokines in vitro.docx

To understand the effect of Seneca valley virus (SVV) VP4 protein on innate immune factors, the VP4 gene was cloned into the pEGFP-C1 expression plasmid to construct the pEGFP-C1-VP4 recombinant plasmid. After the recombinant plasmid was transfected into 293 T cells, the cell fluid was collected 24 h after transfection for western blot assay to identify the correctness of VP4 protein expression. Cell culture medium was collected from un-transfected and transfected cells at three time points (12, 24, and 36 h). mRNA expression levels of cytokines (IL-1α, IL-1β, CCL-2, CCL-5, CXCL-10, and TNF-α) at three time points were detected by quantitative real-time PCR (qPCR) method, and relative quantitative analysis was performed by 2-ΔΔCt method. The results indicated that the expressed SVV VP4 protein exhibits good activity in vitro. Overexpression of the VP4 protein could significantly promote the transcription of IL-1α and IL-1β at 24 and 36 h. In addition, the transcription of CCL-2 and CCL-5 was also significantly promoted at 36 h, whereas the transcription of CCL-10 was significantly promoted only at 12 h. The TNF-α transcription was significantly inhibited at all the three time points. This study provides an important basis for the pathogenic mechanism of SVV and vaccine design in the future.