Molecular Mechanisms Of IL-10 Up-regulation In PAM Induced By Nucleocapsid Protein Of PRRSV

Porcine reproductive and respiratory syndrome(PRRS), also called "blue ears disease", is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus. PRRS is one of the most important diseases constantly bringing huge economic losses to world swine industry. The typical characteristic of PRRSV infection is immunosuppression, including weak innate immune response and inadequate adaptive immune response, especially poor induction of neutralizing antibodies, leading to co-infection and secondary infection with other viruses and bacteria.Cytokines are certain proteins or small peptides delivering messages between cells or inside a single cell, many of which are of great importance in both innate immune response and adaptive immune response. Interleukin-10(IL-10) is a pluripotential cytokine with immunosuppressive properties. Several virus infections which can cause immunosuppression of swine come along with prominent up-regulation of IL-10, among which is the porcine reproductive and respiratory syndrome virus(PRRSV). Our research revealed that nucleocapsid protein of PRRSV ZCYZ strain could significantly up-regulate IL-10 mRNA of PAM(ATCC,3D4/2). Several experiments were conducted to elaborate the molecular mechanisms:1. Construction and identification of PRRSV(ZCYZ) nucleocapsid protein eukaryotic expression vectorSpecific primers with Flag tag in N-terminal were designed according to nucleocapsid protein gene sequence of PRRSV(ZCYZ). pCI-neo-N was constructed through double enzyme digestion of target gene and empty vector. After PCR, double enzyme digestion and gene sequencing verification of recombinant plasmid, IFA and Western-blot were carried out to test the expression property of pCI-neo-N. Results showed that the recombinant pCI-neo-N with expression property was successfully constructed.2. Establishment of a SYBR Green Ⅰ-based realative quantative real-time PCR for detection of porcine IL-10We designed two pairs of real-time PCR primers for the amplification of porcine IL-10 and β-actin, constructed standard plasmid recombinants carrying the sequence of these two genes respectively, and finally established a SYBR Green Ⅰ-based 2-△△Ct real-time PCR detection system. Results revealed that this system showed good linear relationship, high sensitivity with a detection limit of 1.0×101 copies/μL, strong specificity and good repeatability. The real-time PCR products showed single meilting peak, and the coefficient of variation for intra-and inter-assay was less than 3 percent. More important, the PCR efficiencies of IL-10 and β-actin were both close to 100%(92.7%、97.8% respectively). In conclusion, this method can be applied in the mRNA analysis of IL-10 expression.3. Molecular mechanisms of PRRSV(ZCYZ strain) nucleocapsid protein on IL-10 up-regulation in PAMWe found PRRSV(ZCYZ strain) nucleocapsid protein can induce significant expression of IL-10, however, the molecular mechanism of IL-10 up-regulation induced by N protein remains unknown so far. In this study, the expression kinetics of IL-10 induced by N protein in both time- and dose-dependent manner were analyzed. The molecular mechanism of increased IL-10 induced by N protein was preliminarily elucidated by investigating related signal pathways involved in MAPK, PI3K, NF-κB and etc. Protein analytical techniques were applied to identify the key amino acid regions and sites of N protein inducing host IL-10 secretion. However, all the deletion mutants remarkably lost the capability of IL-10 up-regulation, indicating the integrality of N protein was essential for IL-10 up-regulation. Besides, several deletion mutants, which could weakly induce IL-10, were analysed for signal pathways to further verify the molecular mechanism of N protein on IL-10 secretion. This study is of great significance to expound PRRSV immunosuppressive mechanism and develop novel vaccines for PRRS prevention.