| Porcine Reproductive and Respiratory Syndrome(PRRS)is an acute,highly contagious disease of pigs caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV).It causes severe immunosuppression and abnormal cytokine expression is one of its key features.In this study,specific primers and Taq Man probes were designed based on the previously obtained porcine IL-18 BP gene sequence.Withβ-actin as the internal reference gene,a real-time fluorescence quantitative PCR method for detecting IL-18 BP expression was established by optimizing the reaction system and procedure.The method was then used to assay the expression of IL-18BP in the sera of pigs before and after clinical immunisation with live PRRSV vaccine.The method was then applied at the cellular level to detect the expression of IL-18BP in PBMC cells vaccinated with strong and weak PRRSV.The results showed that the linear relationship was good when the concentration of IL-18BP andβ-actin positive plasmid standard was 1×10~8～1×10~4copies/μL,and the correlation coefficients were R~2=0.998 and R~2=0.996.The sensitivity results showed that the minimum detection limit of this method was 10 copies/μL,which was 1000 times higher than that of ordinary PCR detection technology.The specificity test results showed that the method had no cross reaction with IL-4 and IFN-γ.Results from clinical serum samples showed that live PRRSV vaccination had an effect on IL-18BP expression,which was elevated at 21 days post-immunisation compared to pre-immunisation.At the cellular level,the relative expression of IL-18BP in PBMC cells following strong and weak PRRSV vaccination was calculated and found to be statistically significant at 3 h,6 h,12 h and 24 h after vaccination,with changes in the relative expression of IL-18BP in the vaccinated group compared to the control group.The relative expression of IL-18BP was higher in the weakly inoculated group compared to the strongly inoculated group.The method developed in this study can be used to directly measure the expression of IL-18BP in samples and also to derive the relative expression of IL-18BP in samples usingβ-actin as an internal reference gene,which can provide technical support for the study of IL-18BP of porcine origin and also provide a theoretical basis for the study of the cellular immune response mechanism mediated by PRRSV when it infects the host. |
