山羊IFN-γ和TNF-α基因SYBR GreenⅠ实时荧光定量RT-PCR检测方法的建立及应用

Establishment and application of SYBR Green Ⅰ real-time fluorescence quantification RT-PCR for IFN-γ and TNF-α genes of goat

根据GenBank中山羊γ-干扰素(IFN-γ)和肿瘤坏死因子-α(TNF-α)的基因序列,设计特异引物扩增目的基因。将IFN-γ、TNF-α基因克隆至p MD-19-T载体,得到各自阳性克隆质粒,以2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。应用所建立的方法检测刀豆蛋白A(Con A)刺激健康山羊外周血单核细胞(PBMC)后不同时间点IFN-γ、TNF-α基因转录量。结果表明,当质粒标准品稀释度为1×10~9copies/L^1×10~5copies/L时,扩增曲线的Ct值与浓度间具有良好的线性关系,相关系数分别为0.999和1.000;熔解曲线分析表明,产物为特异性单峰且重复性较好。IFN-γ在第2小时和第48小时这两个时间点出现mRNA转录高峰,在第2小时检测到IFN-γ转录量为1.26×10~5copies/L,第48小时检测到转录量为8.56×10~4copies/L;TNF-α在0~24 h呈现下调的表达趋势,第48小时出现mRNA转录高峰,转录量为2.73×10~5copies/L;研究结果将为IFN-γ、TNF-α的定量分析提供技术平台。

Interferon-γ（IFN-γ） and tumor necrosis factor-α（TNF-α） of goat were selected according to GenBank to design specific primers to amplify the target genes.The two kinds of genes were cloned into p MD-19-T vector to obtain the respective positive clone plasmids,which were used as standards to create a standard curve.Melting curve analysis as well as sensitivity,specificity and reproducibility experiments were conducted.With the proposed method,the gene transcription levels of IFN-γ and TNF-αat different time points were detected through the established method,after concanavalin A（Con A）stimulated peripheral blood mononuclear cells（PBMC） in healthy goat.The results showed that,there was a good linear relationship between the Ctvalue and the concentration of the amplification curve when the dilution of plasmid was 1 ×10^9 copies/L ～1 ×10^5 copies/L,and the correlation coefficient was≥-0.996.The melting curve analysis showed that the product was specific and reproducible.The m R-NA transcription peak of IFN-γ occurred at 2 h and 48 h,and IFN-γ transcription was 1.26×10^5 copies/L at 2 h and 8.56×10^4 copies/L at 48 h.The mRNA transcription of TNF-α showed a decline trend in 0 h～24 h,and the peak occurred at 48 h.The transcription amount was 2.73×10^5 copies/L.The results would provide a technical platform for the quantitative analysis of IFN-γ and TNF-α.