Detection Of Porcine Cytokines And Its Preliminary Application

Cytokines are secreted by stimulated immune cells and non-immune cells with broad biological activity. Detection of cytokines can be used for diagnosis of a variety of diseases (infection, inflammation, auto-immune disease, etc.) and evaluation of cellular immune function after vaccination. It's also important for the study of interactions between the virus and host immune system.Cytokines are a large body of functional groups. There're many ways for cytokines'detection. According to test theory and techniques, detection techniques of cytokines can be divided into immunological detection methods, biological methods and molecular biological methods. At present, the most commonly used methods include enzyme-linked immunosorbent spot test (ELISPOT), quantitative RT-PCR, intracellular cytokine detection, enzyme-linked immunosorbent assay (ELISA) and so on.ELISPOT technology is established based on the hemolytic plaque technique and ELISA techniques. ELISPOT could detect cytokines on the single cell level precisely. Currently ELISPOT technology in the world is not yet standardized. Researchers must use the best match of the capture antibody and detection antibody, ELISPOT plates, the substrate and time. So in practice various factors needed to be optimized in order to build a stable detection system.Quantitative PCR technique is based on real-time detection of fluorescent signals and could make accurate quantitative analysis. Currently in pathogen detection this technique has been widely used. This technique includes SYBR Green I and TaqMan probe. SYBR Green I could bind double-stranded DNA non-specificly. This method is suitable for all primers. It's simple, low,costing. However, since it can bind dsDNA, primer dimers or non-specific amplified product could bind with it. Therefore, melting curve analysis is needed to remove the influence. The TaqMan probe method is to add TaqMan probe into the PCR reaction system. This method requires good primer design. The specificity of detection is high, and the results are more reliable.Recently, cellular immunity of pigs gradually attracted people's attention. Pigs are important economic animals. Pigs are vulnerable to a variety of infectious diseases including swine fever, porcine reproductive and respiratory syndrome (PRRS) and so on. These diseases caused great economic losses to the pig industry worldwide. Studies on interaction between the virus and pig immune system, especially the cellular immune response mechanisms are helpful for preventing and treating viral disease, also helpful for the evaluation of new vaccine. In addition, pigs are important experimental animals. Pigs are similar with humans in anatomy, physiology and biochemistry, and many other aspects. Pigs are considered to be the most appropriate animal models for the study of human diseases. Pigs as experimental animals have been often used as models of xenograft rejection, therefore studies on the immune system and immune responses are significant in this field.In this study, we focused on studies on the detection of porcine cytokines. We used Wuzhishan miniature pigs in this experiment, and established detection methods of porcine cytokines, including ELISPOT detection for porcine IFN-y and real-time quantitative RT-PCR methods for porcine IL-2, IL-4, IL-10.To establish ELISPOT detection method for detecting porcine IFN-γ, we selected the PVDF membrane 96-well plates, and then optimized capture antibody and detection antibody concentration, cell number in the plate and virus concentration. The results showed that when the capture antibody and detection antibody concentrations were 1:60, cell number in the plate was 2×10⁵ cells/well, stimulating virus load was 2×10⁵ TCID₅₀. We established a stable detection system.To establish real-time quantitative RT-PCR detection method for detecting porcine IL-2, IL-4, IL-10, we used miniature pig cDNA as the template to amplify the conserved region of porcine IL-2, IL-4, IL-10 gene, and then the genes was cloned into the T vector to construct the corresponding recombinant plasmid. Then the recombinant plasmids were 10-fold gradient diluted, and used as plasmid standards for constructing calibration curve. The results showed that the standard curve correlation coefficients were greater than 0.990, and the amplification efficiency was 90% to 105%. The method of the detection was then evaluated. The results showed that the detection method was with high sensitivity, the detection limit was 10²copies/μL. Repeatability was good. The coefficient of variation was lower than 5%. The evaluation results further demonstrated that the established quantitative RT-PCR detection method was reliable.On the basis of establishing these detection methods, we conducted a preliminary application. We used ELISPOT method to detect IFN-y on 0,7,14,21 days after PRRSV inoculation. The results showed that after inoculation, the expression of porcine IFN-y showed a clear upward trend, on 21 days after inoculation IFN-y reached the peak. Pigs that were not inoculated showed no upward trend in expression level of IFN-y. This indicated that pigs infected with highly pathogenic PRRSV could secret higher IFN-y. Highly pathogenic PRRSV could induce effecient T cell pigs in pigs.We also analyzed IL-2, IL-4 and IL-10 mRNA expression after innoculation. IL-2, IL-4, IL-10 mRNA expression in pigs inoculated with highly pathogenic PRRSV with quantitative Taqman PCR, whileβ-actin was used as internal control. Double standard curve method was used for relative quantification, according to the measured Ct value, toβ-actin as internal reference. The results showed after inoculation cytokines in some pigs changed a lot, while cytokines in the control group changed a little comparatively. In the further study samples need to be increased so that regularity could be summed up.This study will contribute to the study of viral infection immune response of pigs, and will also be helpful for comparative medical research of pigs as experimental animals.