Preliminary Study On Anti-inflammatory And Anti-viral Effects Of Viperin Gene In Pig

Viperin is localized in the endoplasmic reticulum and lipid droplets of cells and has low expression levels at normal.It can be induced by IFN,LPS,poly(I:C),and multiple viruses in a variety of cells.It plays an important role in the body's resistance to pathogens.At present,the anti-inflammatory and antiviral effects of Viperin gene are mainly concentrated in the research of human diseases.There is less research about the role of Viperin gene in pigs.In this study,the Viperin eukaryotic expression porcine PB-Viperin was transfected into PK15 cells,and the Viperin gene overexpression PK15 cell line was screened by fluorescence microscopy,real-time fluorescence quantitative PCR and Western blot methods respectively;In order to investigate the anti-inflammatory effect of porcine Viperin,LPS treatment of PK15 cells as an inflammatory cell model,RT-PCR and Western blot analysis of TLR4 / NF-?B,JAK / STAT signaling key genes m RNA and protein expression levels,to evaluate the Anti-inflammatory effect of overexpression of porcine Viperin gene at the cellular level;Further study was conducted on the antiviral effects of the porcine Viperin gene,using Virusin-overexpressing PK15 cells infected with CSFV as the study object,from virus titer detection,immunofluorescence assay and Western blot protein level detection to test the effect of porcine viperin gene on CSFV replication,and the expression of IRF3 and STAT1 was detected by RT-PCR and Western blot,which revealed the molecular mechanism of porcine Viperin gene anti-CSFV.The result is as follows:The construction of the porcine Viperin gene overexpression cell line: the result of fluorescence microscopy showed that both PB-Viperin and PB transfected PK15 cells emitted green fluorescence,indicating that the vector successfully transfected PK15 cells;RT-PCR and western blot results show that the expression of Viperin m RNA and protein in the cells of PB-Viperin transfection group was significantly higher than that of PB-Viperin transfection group,which showed porcine Viperin gene was finished overexpressed in PK15 cells,and a stable PK15 cell line with overexpression of Viperin gene was obtained by purine mycin screening.The function of Anti-inflammatory of porcine Viperin gene: LPS increased the expression levels of key genes in TLR4/NF-?B and JAK/STAT pathways,that is,LPS activated TLR4/NF-?B and JAK/STAT signaling pathways;The results of RT-PCR showed that PK-PBV cell was lower than IL-6 m RNA of PK-PB cells,the m RNA expression of PK-PBV cell group was upregulation,and the difference was significant.In the control group,My D88?TBK1?NF-?B?IRF3 and IFN? gene,m RNA expression in PK-PB and PK-PBV were not significantly different.After LPS treatment,overexpression of Viperin can down-regulate the expression of My D88?TBK1?NF-?B?IRF3 and IFN? gene m RNA in TLR4 signaling pathway with different degrees.The decrease of m RNA expression of TBK1,and IFN? was significantly different.The results suggest that Viperin may inhibit cellular inflammatory response through TLR4 signaling pathway.LPS could increase the expression of STAT1 m RNA and protein in PK-PB and PK-PBV.Compared with PK-PB,the expression of STAT1 m RNA and protein in PK-PBV of PK-PB group decreased.In the LPS-activated TLR4 signaling pathway,the PK-PBV cell group can down-regulate and the levels of Viperin m RNA and protein were also downregulated.Results suggest that overexpression of Viperin can decrease the expression of STAT1 m RNA and protein in LPS.After AG490 inhibited the jak-stat pathway,the expression of Viperin protein decreased significantly,while the expression level of Viperin protein increased significantly.Compared with LPS group,the expression of STAT1 m RNA and protein decreased in group of L+A,but the expression of viperin m RNA and protein increased.Compared with PK-PB cells,the expression of STAT1 m RNA and protein in PK-PBV decreased significantly;The expression level of Viperin m RNA and protein was significantly increased.It is suggested that Viperin may exert an anti-inflammatory effect through the regulation of TLR4 /JAK / STAT pathways.Virus antiviral effect of porcine Viperin gene: Compared with PK-PB cells,after CSFV infection,the titer of the virus decreased obviously,and the decrease was related to the processing time of CSFV and the complex number of infection.The expression of E2 protein was significantly increased after CSFV infection.Compared with PK-PB cells,the expression of E2 protein in PK-PBV cells decreased significantly compared with that of PK-PB cells.It was suggested that Viperin overexpression could inhibit the expression of CSFV E2 protein,suggesting that Viperin could inhibit the replication of CSFV.m RNA expression of IRF3 and STAT1 was up-regulated in PK-PBV cells after CSFV infection,and IRF3 and STAT1 m RNA expression was up-regulated in PK-PBV cells after stat signal was blocked.The results suggest that the inhibition of csfv replication by porcine Viperin may regulate the expression of IRF3 through JAK/STAT pathway.