鸡细胞因子实时荧光定量PCR检测方法的建立及应用

Establishment and application of a quantitative real-time PCR assay for detection of chickens' cytokines

根据鸡IL-6I、L-1β、IFN-γ、TGF-β4 mRNA的保守序列设计特异性引物,提取经鞭毛蛋白刺激的鸡巨噬细胞HD11总RNA,并反转录成cDNA,建立了实时荧光定量PCR方法。结果显示,融解曲线均呈单一峰;同一份cDNA经过3个相同反应体系的实时荧光定量PCR扩增后,其扩增曲线基本重合。HD11细胞经鞭毛蛋白刺激后,前炎性细胞因子IL-6和IL-1β的相对表达量分别为14.90和29.09,与对照组相比显著升高(P<0.05);而IFN-γ与抗炎性细胞因子TGF-β4的相对表达量无显著变化(P>0.05)。应用该方法检测了3份沙门菌感染鸡血液样品异嗜白细胞中这4种细胞因子的表达水平,前炎性细胞因子IL-6、IL-1β的相对表达量分别为6.19和2.39,与对照组相比显著升高(P<0.05);TGF-β4的相对表达量无明显变化(P>0.05)。结果表明,建立的实时荧光定量PCR方法特异性强、重复性好,为定量检测鸡细胞因子的一种快速、准确的方法。

The specific primers were designed according to the conserved sequences of IL-6,IL-1β,IFN-γ,and TGF-β4 mRNAs,respectively.Total RNA was extracted from chicken macrophage cell HD11 stimulated by flagellin,then it was reverse-transcribed into cDNA.A quantitative real-time PCR（QRT-PCR） method was established to evaluate the relative expression levels of the four cytokines,respectively.There was a single peak in the melting curve,and the amplification curves of the same cDNA were superpositions in teraplicate.Stimulating by flagellin,the relative expression levels of inflammatory IL-6 and IL-1β were 14.90 and 29.09 in HD11 cells respectively and were higher than that of the controls（P0.05）,while the relative expression level of IFN-γ and anti-inflammatory TGF-β4 didn＇t change significantly in HD11 cells（P0.05）.The expression levels of inflammatory IL-6 and IL-1β were detected to be 6.19 and 2.39 respectively,in three chickens＇ heterophils infected by Salmonella by this method and were higher than that of the controls（P0.05）,while the relative expression level of TGF-β4 didn＇t change significantly（P0.05）.The results showed that the established QRT-PCR was specific and reproducible,and could be used for the rapid and accurate detection of chicken cytokines.