犬源干扰素刺激基因ISG15和IFIT2荧光定量PCR检测方法的建立及应用  

Development and application of SYBR GreenⅠreal-time PCR for detection and quantitation of canine ISG15 and IFIT2

作　　者：王改丽 程悦宁[1] 张淑琴[1] 王建科[1] 王超[1] 孙娜[1] 易立[1] 程世鹏[1] WANG Gai-li;CHENG Yue-ning;ZHANG Shu-qin;WANG Jian-ke;WANG Chao;SUN Na;YI Li;CHENG Shi-peng(Key Laboratory of Special Animal Epidemic Disease,Ministry of Agriculture,Institute of Special Economic Animals and Plants,Chinese Academy of Agricultural Sciences,Changchun 130112,China;Jilin Province Animal Husbandry and Veterinary Academy of Sciences,Changchun 130062,China)

机构地区：[1]中国农业科学院特产研究所,农业部经济动物疫病重点实验室,吉林长春130112 [2]吉林省畜牧兽医科学研究院,吉林长春130062

出　　处：《中国兽医学报》2020年第6期1119-1123,1135,共6页Chinese Journal of Veterinary Science

基　　金：中国农业科学院创新工程基金资助项目(20140204066NY);中央级公益性科研院所基本科研业务费专项资助项目(1610342017022)。

摘　　要：干扰素刺激基因在抗病毒及调节IFN信号通路的过程中具有重要作用。本课题组前期对犬瘟热病毒(CDV)感染宿主前后全转录组进行测序分析,发现干扰素刺激基因ISG15和IFIT2明显上调,为了进一步探究ISG15和IFIT2对CDV感染的影响,本研究设计合成了犬源ISG15、IFIT2和持家基因GAPDH的特异性引物,分别进行PCR扩增,构建质粒标准品,建立荧光定量PCR检测方法,并对该方法的特异性、灵敏性和重复性进行检验。结果表明,犬源ISG15、IFIT2和GAPDH的荧光定量PCR标准曲线的相关性R^2均大于0.98,表明线性关系良好。ISG15、IFIT2和GAPDH的融解曲线均为单峰,扩增产物单一,且峰值一致,表明建立的荧光定量PCR方法特异性良好。组内与组间的变异系数均小于0.2%,表明该方法重复性较好。对CDV感染宿主细胞前后的ISG15和IFIT2 mRNA表达水平进行检测,发现CDV感染后ISG15和IFIT2的表达量升高,表明ISG15和IFIT2在CDV感染过程中发挥重要作用。该结果为针对CDV与干扰素抗病毒研究的开展提供重要的技术手段,并为进一步揭示CDV致病的分子机制奠定基础。The interferon-stimulating genes play an important role in antiviral and IFN signaling pathways.The previous study had found that ISG15 and IFIT2 expression were increased in CDV-infection cells by sequencing whole transcriptome.In order to further explore the effect of ISG15 and IFIT2 on CVD infection,the gene sequences of ISG15,IFIT2 and GAPDH were amplified and fluorescence quantitative PCR methods were established with canine ISG15,IFIT2 and GAPDH,and then the repeatability,sensitivity and specificity of real-time PCR were detected.The results showed a precise linear relationship with a correlation coefficient R^2>0.98.Dissolve curve were specific narrow single melting peak and reproductive.The variation coefficients were less than 0.2%within and between assays.The mRNA expression of ISG15 and IFIT2 was detected in CDV-infection cells,and the result showed that the expression of ISG15 and IFIT2 was increased after CDV infection,indicating that ISG15 and IFIT2 played an important role in the process of CDV infection.The above results will provide an important technical instrument for the study of CDV and interferon antiviral,and lay a foundation for further revealing the molecular mechanism of CDV disease.

关 键 词：犬瘟热病毒 干扰素刺激基因 ISG15 IFIT2 荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]