【摘要】 为了建立并应用鸡相关免疫抑制性受体及其配体的检测方法,试验根据GenBank中程序性死亡因子-1(PD-1)及其配体PD-Ls(PD-L1、PD-L2)的基因序列分别设计特异性引物,建立这3种基因的SYBR GreenⅠ实时荧光定量RT-PCR检测方法,并对鸡法氏囊病毒(IBDV)感染雏鸡7 d的外周血单核细胞(PBMC)中3种基因mRNA表达水平进行检测。结果表明:建立的检测方法在1×101~1×108copies/μL模板范围内具有良好的线性关系,相关系数均大于0.990,重复性试验的组内及组间变异系数均小于3%;应用所建立的方法对临床样品进行检测,IBDV感染组PBMC中PD-1、PD-L1和PD-L2 mRNA表达量与对照组相比均显著升高(P<0.05)。说明所建立的检测方法具有良好的敏感性、特异性和重复性。更多还原

【Abstract】 To establish and apply a method for detecting of an immunesuppression-associated receptor and its ligands in chickens,the specific primers were designed according to the gene sequences of the programmed death-1(PD-1) and its ligands(PD-L1 and PD-L2) published in the Genbank,a SYBR Green Ⅰ real-time fluorescence quantitative RT-PCR assay was established to detect the mRNA expression levels of these three genes in chicken peripheral blood mononuclear cells(PBMC) on the 7th day after infectious bursal disease virus(IBDV)infection. The results showed that the established method had a linear dynamic range from 1 × 101 copies/μL to 1 × 108 copies/μL,and all the correlation coefficients were greater than 0. 990,and the intra-and inter-assay coefficients of variation in the repeatability tests were less than3%. Further more,the results of clinical sample detection also demonstrated the mRNA expression levels of PD-1,PD-L1 and PD-L2 in the IBDV-infected group were significantly increased(P < 0. 05) compared with the control group,respectively. The results indicate that the method has good specificity,sensitivity and repeatability.更多还原