3种猪细胞因子mRNA实时荧光定量RT-PCR检测方法的建立与应用

Establishment and Application of Real-time Fluorescent Quantitative RT-PCR Method of the Detection of Three Swine Cytokines

为建立猪细胞因子SYBR GreenⅠ实时荧光定量RT-PCR检测方法,根据GenBank中3种重要的猪细胞因子即猪白细胞介素-2（interleukin-2,IL-2）、α-干扰素（interferonα,IFN-α）和肿瘤坏死因子-α（tumor necrosis factor,TNF-α）的基因序列,设计特异引物扩增目的基因。将3种基因克隆至pMD18-T载体上,得到各自阳性克隆质粒,以3种阳性质粒为标准品建立标准曲线并进行熔解曲线分析以及灵敏性、特异性和重复性试验。结果表明,当标准品稀释度为1×101~1×106拷贝/μL时,3种基因的Ct值与浓度间具有良好的线性关系,相关系数均≥0.992。熔解曲线分析表明,产物为特异性单峰且重复性较好。应用建立的方法对猪繁殖与呼吸综合征病毒（PRRSV）TJM-F92株免疫的30日龄猪外周血单核细胞（PBMC）中IL-12、IFN-α和TNF-α表达量进行检测,结果发现,免疫了PRRSV TJM-F92株的猪PBMC细胞内3种细胞因子表达量均极显著升高（P〈0.01）。研究结果为IL-12、IFN-α和TNF-α的定量分析提供了技术平台。

In order to establish the assay of SYBR GreenⅠ Real-time fluorescent quantitative RTPCR for detecting three important swine cytokines IL-12,IFN-αand TNF-α.Three pairs of specific primers were designed according to the sequence of IL-12,IFN-αand TNF-αfrom GenBank to amplify the objective genes.The three genes were respectively cloned to the pMD18-T vector.The corresponding plasmids were identified by sequencing,and they were then used as quantitative template to construct the standard curve and to analyze the melting curve,detection sensitivity,specificity and repeatability.The results showed that Ct value of the three genes had good linear relationship（R2≥0.992）with the dilution ranging from 1×101 to 1×106copies/μL.The melting curve displayed a single peak and good repeatability.The established assay was used to detect the transcriptional level of IL-12,IFN-αand TNF-αmRNA in the swine PBMC inoculated PRRSV TJMF92.The results indicated that mRNA levels of IL-12,IFN-αand TNF-αin the swine PBMC inoculatedPRRSV TJM-F92 were all significantly increased（P〈0.01）.The research provides the platform for quantitative analysis of swine cytokine IL-12,IFN-αand TNF-α.