羊凋亡细胞因子Fas、FasL、TNFR1和TNF-α实时荧光定量检测方法的建立

Development of a SYBR Green Ⅰ Real-time PCR Method for Detection of Sheepapoptotic cytokines

为探讨羊痘病毒感染后机体的凋亡细胞因子mRNA表达水平的变化,深入探讨羊痘病毒免疫机制,本研究设计针对羊凋亡细胞因子Fas、FasL、TNFR1和TNF-α特异性引物,建立羊凋亡细胞因子实时荧光定量方法。分别建立荧光定量PCR反应体系并进行优化。结果表明:Fas、FasL、TNFR1和TNF-α的SYBR Green Ⅰ实时荧光定量PCR各基因的溶解曲线均呈单一溶解峰。组内变异系数均小于2.00%。本研究建立的羊凋亡细胞因子SYBR Green Ⅰ荧光定量RT-PCR检测方法为凋亡细胞因子mRNA表达水平提供了技术平台。

For detection of the related apoptotic cytokines in Sheep,the R eal-time fluorescencequantitative PCR method were designedaccording to t he sequence of the Sheepapoptotic cytokines（Fas, FasL, TNFR1 and TNF-α）genes.The conserved region of the S heep apoptotic cytokineswer eamplifiedby PCR and cloned in top MD-18T vector.The recombinant plasmidplasmid DNA were used astemplate to optimize assay condition of developing a SYBR Green I real-time PCR for detectio n.The results showedthat theCtofapoptotic cytokines genes had a good linear relationshipwiththem elting curve showeda single peak . The develo pedrea - ltime PCR assay could quickly detec tapoptotic cytokines genes in expansion range with high effciency,thus providing the basis forquantitative analysis of apoptotic cytokines gene expression.