【摘要】 为测定鸡TBK1(chTBK1)基因在家禽抗感染天然免疫中mRNA表达水平变化情况,本研究根据chTBK1序列,设计3对特异性引物,基于chTBK1和β-actin双标准曲线建立了荧光定量PCR(qRT-PCR)检测方法,并测定在禽流感病毒刺激下鸡成纤维细胞(DF-1)中chTBK1表达量的变化情况。结果表明,建立的标准曲线Ct值与模板浓度的对数呈现良好的线性关系(R~2均大于0.99),能特异地检测chTBK1,检测灵敏度可达59拷贝数/μL,且组内及组间变异系数小于2%;chTBK1在DF-1细胞中的mRNA水平在病毒刺激下呈现上升趋势。该qRT-PCR检测方法具有良好的特异性、灵敏性和重复性,为研究chTBK1在鸡抗病毒感染中的作用提供了有效工具。更多还原

【Abstract】 In order to determine the change of mRNA level of chicken TBK1(chTBK1) gene in poultry antiviral infection,specific primers were designed based on the sequence of chTBK1 and the fluorescence quantitative real-time PCR(qRT-PCR) was established based on the double-standard curve of reference gene β-actin and target gene chTBK1.Also the expression of chTBK1 in chicken embryonic-fibroblast cell line(DF-1) under the stimulation of avian influenza virus was determined.The results showed that the standard curve has a good linear relationship between Ct values and the logarithm of template copy number,with an excellent correlation coefficient(R~2>0.99).The method can specifically detect chTBK1,the detection threshold is 59 copies/μL and the intra and inter group coefficients of variation are less than 2%.The expression of chTBK1 showed a rising trend under the stimulation of the influenza virus.The method has good specificity,sensitivity and repeatability,thus providing an effective tool for further research on the role of chTBK1 gene in chicken antiviral infection.更多还原