用实时荧光定量RT-PCR方法定量绵羊PrP基因的表达  

Quantification of sheep prion gene expression using real-time RT-PCR

作　　者：韩彩霞[1] 赵德明[1] 吴长德[1] 宁章勇[1] 杨建民[1] 刘美丽[1] 马李颖[1] 

机构地区：[1]国家动物海绵状脑病实验室中国农业大学,北京100094

出　　处：《中国农业大学学报》2006年第2期61-64,共4页Journal of China Agricultural University

基　　金：国家自然科学基金资助项目(3037106230400325);教育部博士点基金资助项目(20020019006)

摘　　要：为快速、准确定量绵羊PrP基因的mRNA,建立了绵羊PrP基因实时荧光定量聚合酶链反应检测方法。根据已报道的绵羊PrP基因序列,设计合成引物;采用RT-PCR方法扩增目的片段;构建标准重组质粒制备标准曲线,用于样品检测。结果发现,中枢神经系统组织PrP基因的表达量(copies/ng总RNA,39420)比外周组织(为7845)的高;在中枢神经系统中,脑干的PrP基因的表达量最高(为67020);外周器官中,淋巴结PrP基因的表达量最高(为29086),肾脏的表达量最低(为125)。建立绵羊PrP基因实时荧光定量PCR方法,对PCR扩增反应中每一个循环的产物进行定量分析,为进一步研究绵羊组织器官的PrP表达在传染性海绵状脑病发生中的作用提供基础数据。The real-time fluorescent quantitative PCR method was established to quantify the sheep prion gene expression. The PrP target fragment was amplified, and the real-time fluorescent RT-PCR technique was used to construct the standard plasmid and curve to quantify samples. Total RNA was isolated from different regions of the central nervous system （CNS） and peripheral organs to quantify the gene expression after the reverse transcript. The results showed that higher mRNA levels were present in CNS than in peripheral organs and the expression levels of PrP mRNA were 39420 copies and 7845 copies per ng total RNA respectively. The highest expression level was found in Obex with 67020 copies per ng total RNA within all CNS examined. In peripheral organs examined, high and low expression was respectively observed in lymph node and kidney, and the expression levels were 29086 copies and 125 copies per ng total RNA. The real-time fluorescent quantitative PCR is currently considered to be the most precise method for nucleic acid quantification and appeared as a powerful tool for further studies on prion diseases pathogenesis.

关 键 词：朊蛋白 RT—PCR 基因表达 实时荧光定量PCR 

分 类 号：S826[农业科学—畜牧学]