Quantitative Detection Of IL-6、IL-18and IFN-γ In SPF Chickens Infected With REV

Interleukin-6, also known as B cell stimulating factor is a necessary factor forinduction of B cells to produce antibodies, the level of its secretion may reflecthumoral immune function. Interleukin-18, also known as IFN-γ inducible factor, is akind of multi-directional and multi-level immunomodulatory cytokine which isclosely associated with the occurrence and development of carcinoma, allergy, auto-immune diseases, and graft-versus-host disease. IFN-γ is a low molecular weightsoluble glycoprotein secreted by activated T lymphocytes and natural killer cells, is animmunomodulatory pleiotropic factor, mammals and birds of IFN-gamma was usedfor infection of the body’s cells mediated immune indicator.Now intensive immunosuppressive disease has brought great harm to the poultryindustry in China. There have been many reports that REV infection can cause seriousimmunosupression of humoral and cell-mediated immune responses. Understandingthe mechanism of immunosuppression and developing strategies to improve theimmune responses of chickens have become the current research hotspots. In thisstudy, immunosuppression caused by REV was used as a model and the role ofchicken IL-18in the immune suppression was discussed. And this study was dividedinto three parts:1. Study on IL-18gene expression in the primary immune organs of SPFchickens infected with REV by real-time quantitative RT-PCR. In order to studyexpression levels of IL-18gene in SPF chickens infected with reticuloendotheliosisvirus,130one day old SPF chickens were randomly divided into2groups. Thetreatment group chickens was infected with REV while the control group wasinoculated with normal saline. Eight chickens of each group were randomly chosenand killed at day7,14,21,28,35,42and49post infection. Spleen, thymus and bursa ofFabricius were sampled for RNA extraction. SYBR GreenⅠ q uantitative real-timePCR method was applied to compare the relative expression quantity of SPF chickensIL-18gene. The results were as follows: IL-18gene expression levels weresignificantly higher (P<0.05) in the spleen,thymus and bursa of Fabricius in SPF chickens from treatment group than those in the control group at day7,14,21,28,35,42and49except the14d in the spleen.2. A real-time reverse-transcription PCR based on SYBR Green I for detectingchicken IL-6and IFN-γ was established successfully. According to the chicken IL-6and IFN-γ gene sequence available in GenBank, a pair of primers was designed fordeveloping a SYBR GreenⅠ quantitative real-time PCR method to detect chicken IL-6and IFN-γ gene while the chicken ribosomal protein subunit4(RPL4) gene was usedas an internal control. To establish the standard curve, the positive plasmid of eachgene served as a standard. The melting curve was also analyzed. The results showedthat the amplification efficiency of this assay was107%, and the linear range was10-2~10-6with good correlation coefficient of0.998, as well as the detection limit reached100copies/μL. It only took4hours from extracting RNA to the end of real-timefluorescent quantitative RT-PCR. The developed real-time PCR assay could quicklydetect IL-6and IFN-γ gene in expansion range with high efficiency, thus providingthe basis for quantitative analysis of IL-6and IFN-γ gene expression.3. Study on IL-6and IFN-γ gene expression in the primary immune organs of SPFchickens infected with REV by real-time quantitative RT-PCR. In order to studyexpression levels of IL-6and IFN-γ gene in SPF chickens infected withreticuloendotheliosis virus,120one day old SPF chickens were randomly divided into2groups. The treatment group chickens was infected with REV while the controlgroup was inoculated with normal saline. Six chickens of each group were randomlychosen and killed at day7,14,21,28,35,42and49post infection. Spleen, thymus andbursa of Fabricius were sampled for RNA extraction. SYBR Green Ⅰ quantitative real-time PCR method was applied to compare the relative expression quantity of SPFchickens IL-6and IFN-γ gene. The results were as follows: IL-6and IFN-γ geneexpression levels were at least significantly higher (P<0.05) at all tested time points inthe spleen,thymus and bursa of Fabricius in SPF chickens from treatment group thanthose in the control group. Our study laid the foundation for investigating themechanism of immunosuppression induced by REV.