山羊IL-1β IL-8和Mx1因子实时荧光定量检测方法的建立

Development of real-time RT-PCR assay based on SYBR Green I for detection of IL-1β, IL-8 and Mx1 Factor in Goats

根据GenBank羊炎性细胞因子(IL-1β和IL-8)和Mx1保守序列,设计特异性引物,利用SYBR Green I染料法建立实时荧光定量检测方法。将羊IL-1β、IL-8和Mx1细胞因子基因保守区域克隆到pMD18-T载体,构建标准质粒。分别以标准质粒为模板建立荧光定量PCR反应的标准曲线、熔解曲线。结果表明,羊IL-1β、IL-8和Mx1细胞因子实时荧光定量检测方法Ct值与标准品呈良好的线性关系,R^2均大于0.990,所有稀释度标准品模板出现特异性熔解峰。应用所建立的方法对山羊痘病毒AV41感染山羊外周血淋巴细胞中IL-1β、IL-8和Mx1 mRNA表达水平进行检测,山羊痘病毒AV41可以刺激机体产生较高的炎性细胞因子。建立的羊IL-1β、IL-8和Mx1细胞因子荧光定量检测方法,可以为山羊痘病毒感染后分子免疫机制研究奠定基础。

The main purpose of this study was to establish a real-time fluorescence quantitative PCR method for detection of goat proinflammatory and antiviral Mx1 factor.Specific PCR products of these genes were utilized to construct recombinant plasmids.The recombinant plasmids were used to optimize assay condition of developing a SYBR Green I real-time PCR for detection.These assays were single specific melting peak for every cytokine.These assays were highly reproducible and a coefficient of variation was less than 2 percent for both intra-and inter-assay.The established assays were successfully used to detect IL-1β,IL-8 and Mx1 mRNA expression levels in peripheral blood mononuclear cells(PBMCs)in goat experimentally infected with goatpox virus(AV41 strain).AV41 strain stimulated the body to produce higher inflammatory cytokines.The establishment of goat IL-1β,IL-8 and Mx1 cytokine fluorescence quantitative detection methods lay the foundation for goat pox virus infection molecular mechanisms.