Influence On The Expression Of TLR3 And IL-10 By Porcine Reproductive And Repiratory Syndrome Virus Encoding Proteins

Porcine reproductive and respiratory syndrome virus (PRRSV) emerged in Europe and in the US in the early 1990s and has since become a problem to the swine industry worldwide. Accumulating evidence has revealed that PRRSV is able to escape immune responses by a variety of strategies such as inhibiting the expression of inflammatory cytokines, regulating Interleukin-10 production for suppression of the immune response, interfering with the functions of immune cells (macro-phages and dendritic cells) and failing to induce type 1 interferon(IFN-α/β), ultimately resulting in viremia and establishment of persistent infection.To explore the influence on the expression of TLR3 and IL-10 by PRRSV encoding proteins, we constructed the recombinant eukaryotic expression plasmids of some non-structural proteins, and found that Nsp1 and Nsp11 were able to inhibit the TLR3 promoter activity and got a preliminary analysis of its possible mechanisms. Meanwhile, we constructed the recombinant eukaryotic expression plasmids of structural proteins, and observed GP5 and N protein were able to induce IL-10. We also constructed the recombinant eukaryotic expression plasmids of Nsp4, Nsp7 and Nsp12 as DNA vaccines and had a study on their immunological characteristics. The results above provided a foundation for the further study on immunosuppressive mechanisms and screening target antigens of PRRSV.Specific research contents include:1. Influence on the TLR3 promoter activity by three non-structural proteins of PRRSV and study on its mechanismsPRRSV genome contains two open reading frames:ORF1a and ORF1b, which encode 14 non-structural proteins. According to recent studys, some non-structural proteins may play an important role in inhibition of type I interferon, while TLR3 as an important receptor molecular can originate the innate immune response by inducing type I interferon and pro-inflammatory cytokines after stimulated by its ligand dsRNA. In order to study the influence on the TLR3 promoter activity by non-structural proteins, the recombinant eukaryotic expression plasmids of some non-structural proteins(Nspl, Nsp1α, Nsp1α-A14. Nsp1β. Nsp7, Nsp11) of PRRSV strain BB0907 were constructed, and these non-structural proteins’ influence on the TLR3 promoter activity was detected by the Dual luciferase report system. The result indicated that Nsp1, Nsp1α, Nsp1β, Nsp11 are able to inhibit the TLR3 promoter activity and the C-terminal zinc finger motif of Nsp1α was essential for its suppression, while the suppression of Nsp11 was dose dependent. In a further study, we found that type I interferon were also able to induce the TLR3 promoter activity, and Nsp11 could inhibite the IFN-β-stimulated TLR3 promoter activity, which suggested that Nsp11 may have the ability to inhibite the JAK-STAT pathway. The results of our study partly explained the reason why PRRSV could trigger a low innate immune response and developed a new direction for studying the immunosuppressive mechanisms of Nsp11.2. Eukaryotic expression of the structural proteins of PRRSV and their influence on the IL-10 promoter activityPRRSV is a member of nonsegmented enveloped viruses with positive-sense (+) RNA genomes, and the ORFs2-7 in its genomes can encode 9 structural proteins. The recent research indicates that PRRSV are able to inhibite the innate immune response and cause a persistent infection by inducing IL-10, while the structural proteins of PRRSV have been proved to play a role in this process. In this study, to explore influence on the IL-10 by structural proteins, the recombinant eukaryotic expression plasmids of some structural proteins of PRRSV strain BB0907 were constructed and identified in BHK-21 cells by IFA. Then these structural proteins’influence on the IL-10 promoter activity was detected by the Dual luciferase report system in PAM(3D4/2) cells. The results indicated that GP5 and N protein can activate the IL-10 promoter significantly, which made a foundation for the further study on its mechanisms of PRRSV inducing IL-103. Construction and immunogenicity of gene vaccines of non-structural protein 4, 7 and 12 of PRRSVPorcine reproductive and respiratory syndrome(PRRS) is a a highly contagious disease of pigs,which is caused by PRRSV. To get a better control of PRRSV, the idea of screening more effective target antigens to develop new types of PRRSV vaccine is always under study. Howere, at the present stage we often focus on analysing the feasibility of structural protein of PRRSV as the immune protective antige with litter reserch about nonstructural protein. Therefore, in order to explore the feasibility of PRRSV nonstructural protein as immune protective antigen, in this study, we refered to the research results of our laboratory and then chose Nsp4, Nsp7 and Nsp12 as Immune target proteins, which were free of immunosuppression. To guarantee the biological activities of each protein, the eukaryotic expression vector pcDNA3.1 was used for constructing Gene vaccines, with the gene GM-CSF and motif KDEL as an immune-enhancing adjuvant, aimed at studying the immunological characteristics of Nsp4, Nsp7 and Nsp12. The results indicated that the Nsp4, Nsp7 and Nsp12 expressed by eukaryotic expression vector were able to cause some humoral immune response in mice which can be enhanced by GM-CSF gene, but the effect of cellular immune response is not satisfactory. In this study, we explored the immunogenicity of non-structural protein 4,7 and 12 of PRRSV preliminarily, and provided an important basis and a new direction for screening target antigens of PRRSV vaccine.