山羊Th1/Th2细胞因子实时荧光定量检测方法的建立

目的 在mRNA 水平对山羊Th1/Th2型细胞因子进行定量分析,建立山羊Th1/Th2型细胞因子实时荧光定量检测方法。方法 根据GenBank山羊Th1(IL-2和IFN-γ)和Th2(IL-4、IL-6和IL-10)细胞因子基因保守序列设计引物,以标准阳性质粒为模板分别进行荧光定量PCR反应的标准曲线、溶解曲线建立。结果 山羊Th1/Th2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,R²均大于0.985,所有稀释度标准品模板出现特异性熔解峰;利用N1蛋白缺失的重组山羊痘病毒(△N1L株)与山羊痘AV41株感染山羊外周血淋巴细胞进行IL-4和IFN-γ mRNA 表达水平检测,△N1L株与山羊痘AV41株均能能够刺激机体IL-4和IFN-γ细胞因子,但二者差异无统计学意义(t=3.333,P>0.5)。结论 本研究建立山羊Th1/Th2细胞因子实时荧光定量检测,为山羊痘基因工程疫苗的研究奠定基础。

Development of a SYBR GreenⅠReal-time PCR method for detection of goat Th1/Th2 cytokines

This study was to establish a real-time fluorescence quantitative PCR method for detection of goat's Th1/Th2 cytokines.The conserved region of goat's Th1/Th2 cytokines gene were amplified and cloned into pMD18-T vector.The plasmid DNA was used as template to optimize assay condition of developing a SYBR GreenⅠReal-time PCR for Th1/Th2 cytokines detection.The results showed that the Ct value of Th1/Th2 cytokines produced a good linear relationship, which R² were all greater than 0.985.All standards were specific narrow melting peak and reproducetire and specific.The established assays were successfully used to detect IL-4 and IFN-γmRNA expression levels in peripheral blood mononuclear cells (PBMCs) in sheep experimentally infected with recombinant goat poxstrain (△N1L strain) and AV41 strain, both could stimulate Th1/Th2 cytokines with no difference in vivo. In this study, real-time fluorescence quantitative detection of goat Th1/Th2 cytokines was established, which laid the foundation for the research of goat pox genetic engineering vaccine.