Establish Of PRRSV And CSFV Real-time Quantitative PCR Methods And Immunological Study Of PRRSV M Protein Recombinant Plasmid

Porcine reproductive and respiratory syndrome (PRRS) is a new infectiousdisease caused by PRRSV, to sow reproductive disorders, piglets and growing pigsrespiratory disease and high mortality rates as the main feature. Swine fever iscaused by the classical swine fever virus (CSFV), a serious, highly contagiousdisease. Swine fever and porcine reproductive and respiratory syndrome is morecommon diseases in the modern pig industry, especially piglets mixed infectionswith high mortality, harm to the pig industry. So to find a reasonably rapid diagnosticmethods for prevention and control PRRS and swine fever significance.In this study, acute death by nursery pigs on a farm, and through theobservation of clinical symptoms, pathological examination and RT-PCR test to thecomprehensive epidemiological characteristics of the disease, the diagnosis ofclinical symptoms, necropsy pathological changes, as well as laboratory tests andother methods Classical swine fever virus and porcine reproductive and respiratorysyndrome virus mixed infection.Step closer to determining PRRS and CSF mixed the accuracy of the diagnosisof infection by real-time PCR applications in the disease clinical testing in thisexperiment, and the two pairs of primers were designed for of PRRSV ORF5structural proteins and CSFV envelope glycoprotein protein GP5gene Real timePCR applications real-time fluorescence quantitative techniques detect differentsuspected incidence of diseased pig collection. And the optimization of quantitativePCR reaction conditions, draw a SYBR Green Ⅰfluorescent quantitative PCRstandard curve, and specificity, and reproducibility of analysis. The results showthat: the better the linearity of the standard curve, and the PCR amplification efficiency is relatively good, draw the curve of the linear relationship between thecopy number (x) and the cycle threshold (Ct) value expressions are: Ct=-2165×LG x+6.96, CT=-2. The188×LG x+7.13, and the Ct value of the sample canbe read from the instrument; will be substituted into the expression of the Ct value ofthe test sample can be calculated in the initial copy number. Repeatability of testresults show that each amplified error is less than one cycle, showing the real-timePCR detection method having a higher reproducibility, thus ensuring the stabilityand reliability of the detection result in the different samples will be differentdilution standard PRRSV, of CSFV were amplified by PCR, established real-timePCR analysis using statistical software group and the group has good repeatability(P>0.05), correlation coefficients are large at0.99. The negative controlamplification characteristics. The test using the optimized conditions, theamplification products are single peak, Tm value uniform, no non-specificamplification.For the purpose of this study to strengthen the prevention and control of thefuture of PRRSV PRRSV major protective antigen gene ORF6gene molecularadjuvant IL-18, to build PRRSV ORF6gene IL-18co-expression of eukaryoticplasmids, and its immunological study. The results show that: the recombinantplasmid pEGFP-the ORF6and pEGFP-IL18-ORF6immunohistochemistry14dafter the first immunization began to produce specific antibodies, and the antibody levelsupward trend over time, but pEGFP-ORF6and pEGFP-IL18-ORF6two groups betweenproduce antibodies difference was not significant (p>0.05). Neutralizing antibody testresults show that the pEGFP-ORF6only low levels of neutralizing antibodies42days afterthe first immunization. Cellular immunity test results, pEGFP-IL18-ORF6induced Tlymphocyte proliferation and promote secretion of IFN-γ and IL-2’s ability to significantlyabove the pEGFP-ORF6and pEGFP-IL18(p <0.05), indicating that IL-18play the utilityof molecular its adjuvant, can enhance the cellular immune response and better Th1-mediated immune response.