Study On Toll-like Receptors In The Innate Immun Induced By Porcine Parvovirus

Porcine parvovirus(PPV)is a member of parvoviridae,containing a negative single-stranded DNA genome.PPV infection mainly causes reproductive clinical syndrome,including infertility,abortion,stillbirth,neonatal death,and reduced neonatal vitality,causing severe harm to pig industry.Studies have shown that PPV can proliferatein on most porcine primary cells and subcultured cells,and causes the cells to obvious pathological changes,so it is always used as a model virus to explore the mechanism of the virus infection and the antivirus mechanism.Nuclear factor kappa B(NF-?B)has been recognized as an important transcription factor related to many immune and inflammatory responses.Toll like receptor(TLRs)can activate the transcription factors such as NF-?B,TRAF6(TNF receptor associated factor-6)and other genes by the My D88 dependent pathway or the My D88 non-dependent pathway,and induces the synthesis and release of inflammatory cytokines,chemokines and interferon,playing an important role in immune responses.In this study,we used porcine kidney-15(PK-15)cells as a cell model to research the m RNA expression of TLR1~10 after PPV infection,and investigate the effect of PPV on NF-?B signaling pathway and immune response.Furthermore,we analyzed the function of TLR9 in the NF-?B signaling pathway and defined the role of TLRs in the innate immune response after PPV infection.The main contents are as follows:1.Ten pairs of primers were designed according to the TLR1~10 gene in Gen Bank.We used fluorescence quantitative PCR to investigate the transcription pattern of TLR1~10 in PK-15 cells after PPV infection.The results showed that TLR1 were up-regulated in PK-15 cells infected with PPV at 3h post-infection(about 3.1 times as much as the control cells),while other times are lower than normal level.The m RNA expression of TLR3 and TLR7 were lower than the normal level in the early stage of viral infection.In addition,the expression of TLR4 and TLR8 rasied at 3,12 and 36 h after PPV infection and other periods were reduced.Moreover,the m RNA expression levels of TLR2 and TLR9 were significantly increased at 24 h in PK-15 cells after PPV infection and reached the peak levels at 48 h.These results indicated that TLR2 and TLR9 m RNA level were significantly up-regulated in pk-15 cells after PPV infection.2.In order to investigate the effect of PPV on NF-?B signaling pathway and immune response in PK-15,this study detected the expression of My D88,TRAF6,NF-?B and IL-6 by fluorescence quantitative PCR.Meanwhile,the expression of IL-6 was also detected after PPV infection by ELISA.The results showed that the m RNA levels of My D88,IRAK1,TRAF6,TAK1?,I?BKB and NF-?B were significantly increased In the late phase of infection.The levels of m RNA and protein of IL-6 reached the peak levels at 36 h.In all,the above results show that PPV activated the NF-?B signaling pathway and induced the expression of inflammatory factors in PK-15 cells.3.To further understand the role of TLR9 in the innate immune response mediated by TLRs-NF-?B,E6446 dihydrochloride was used to determine the role of TLR9 in NF-?B signaling pathway in PK-15 cells after PPV infection.The results showed that,When its concentration were 0.02?M and 0.03?M,E6446 dihydrochloride could inhibit the expression of TLR9.Meanwhile,the expression of IL-6 was also down-regulated.And the expression of IL-6 achieved the peak at 48 h after trated by E6446 dihydrochloride,while the expression of IL-6 achieved the peak at 24 h in PK-15 cells after PPV infection.The results showed that PPV could activate NF-?B signaling pathway through My D88 dependent pathway,and regulate the expression of inflammatory cytokines IL-6.