Regulation Of The Structural Proteins Of Classical Swine Fever Virus On Innate Immune Response Mediatedby Toll Like Receptors Of Porcine Alveolar Macrophages

Classicial swine fever(CSF),leading to large loss of pig industries, attracted by Classicial swine fever virus(CSFV), is an list A of the Office International des Epizooties(OIE), high contagious, fetal disease of swine worldwide.The innate immune response is thefirst line of defense for invading pathogens. But currently, the mechanism of innate immune response to classical swine fever virus sturctural proteins still remains unclear. The innate immune response is the first line of defense against an invading pathogens. Toll-like receptors(TLRs) are aware of microbial pathogens-associated molecular patterns(PAMPs), activate the expression of interferons, chemotactic factors and inflammatory cytokines and eliminate pathogenic microorganisms. The study about the mechanism between CSFV and TLRs mainly focus on the activation of immune cells and indueⅠinterferon and inflammatory cytokines production. This study analyzes the relationship between the CSFV structural proteins and TLRs(TLR2,TLR3,TLR4 and TLR7) mediated the innate immune signaling pathways, as well as the influence of the expression of the immune effector molecules downstream. In this study, we established over-expressing CSFV structural proteins in macrophages, and determinied the expression of TLRs and immune effector molecules, to understand the influence between CSFV structural proteins and TLRs mediate innate immune responses. The results as below:1. Add the “Flag” tag to the C-terminal by primer design. The genes of CSFV structural proteins(C, Erns(E0), E1 and E2) were cloned by RT-PCR from “pBlueScript ⅡSK-CSFV Shimen”. Inserted them into pCD513 B vector to construct four eukaryotic expression recombinant lentiviral plasmid pCD513B-C-Flag, pCD513B-Erns-Flag, pCD513B-E1-Flag and pCD513B-E2-Flag. The double enzyme identification and sequencing results showed that all of the four eukaryotic expression recombinant lentiviral plasimd were constructured successfully.2. By lentivirus packaging system, the recombinant lentiviral plasimd and packing mixer were transfected into HEK 293 T cells, then collected the recombinant lentiviruses which infected porcine alveolar macrophages. Analyze by fluorescence inverted microscope and Western blot, which showed CSFV structural proteins over-expressing in.macrophages.3. Determined the expression of TLRs by real-time PCR and Western blot. The results indicate that the Core protein up-regulated the expression of TLR2 and TLR3, mainly by TLR2 and TLR3 signaling pathways to activate innate immune response; Erns can up-regulate the expression of TLR3 significantly, promote the production of TRIF and IRF3, inhibit MyD88 production,mainly by TLR3 signaling pathway to activate the innate immune response; E1 and E2 result in a suppression of MyD88, IRF7 and NF-κB, inhibiting the MyD88 mediated TLRs signaling pathways.In summary, we established the macrophage over-expressing CSFV structural proteins in the present study, having a preliminary understanding of the relationship between CSFV structural proteins and TLRs(TLR2, TLR3, TLR4 and TLR7) mediated the innate immune response, ladi the foundation for further study.