鸡体抗病毒蛋白基因荧光定量PCR检测方法的建立和初步应用

Establishment and Preliminary Application of Real-Time Fluorescence Quantitative PCR Assay for Antiviral Protein Genes in Chicken

为建立检测鸡干扰素诱生的3种抗病毒蛋白(IFIT5、PKR和OASL)基因表达水平的荧光定量PCR方法,并将其应用于实践,本研究设计和筛选了3个基因的特异性扩增引物,制备了各基因的质粒标准品,绘制了荧光定量PCR的标准曲线,并检测了该方法的灵敏度、重复性和特异性。结果表明所建立的方法敏感度高、特异性强、检测线性范围广、重复性好,应用该方法检测了新城疫病毒感染SPF鸡胚后相关基因的表达情况,发现在病毒感染后IFIT5、PKR与OASL基因的表达水平均迅速提高。

To develop a fluorescence quantitative PCR method for detecting gene expression levels of three types of interferon- stimulated antiviral proteins,IFIT5,PKR and OASL,specific primers were designed and screened. The PCR products were utilized to construct recombinant plasmids,which served as templates to establish standard curves. Then,the sensitivity,repeatability and specificity of the method were detected. The results showed that the method had high sensitivity and specificity,wide linear range and well repeatability. The related gene expression in specific- pathogen- free（ SPF） chicken embryo after Newcastle disease virus infection indicated that the expression levels of IFIT5、PKR and OASL increased quickly after virus infection.