实时荧光定量PCR构建绵羊PrP基因标准品质粒和标准曲线

羊痒病是一种传染性的致死性神经退行性疾病,常引起绵羊和山羊发病,该病在欧洲流行了大约250年,但是它的流行病学和传播机制还不是很清楚.正常的朊蛋白（PrPc）不能引起神经退行性病变,虽然目前已经对许多组织的PrP mRNA进行了检测,但是对它的生物学功能还知之甚少,对PrP基因表达的机制尚不清楚.研究显示,不同组织来源的细胞中朊蛋白的表达程度差异很大,主要出现于神经细胞中.PrPc在细胞中的高水平表达可促使PrPc向PrPsc转变,目前的研究中,对绵羊PrP mRNA的转录机制尚未阐释清楚.因此,对绵羊外周和中枢系统PrP mRNA的表达进行定量,有助于探讨各组织器官中的PrP在羊痒病的发生过程中的作用.

Construction of Sheep PrP Gene Standard Plasmid DNA and Curve Using Real-time RT-PCR

Here we report the construction of sheep PrP gene standard plasmid DNA and curve using realtime RT-PCR. Total RNA was extracted from each sample and the fragments of target gene were amplified by RT-PCR. The plasmid was constructed for calibrating unknown samples. In this study, the constructed plasmid containing only the target gene was used to construct a calibration curve. The absolute standard curve method was shown to be of high linearity, sensitivity and reproducibility. The purpose of this study is to investigate the quantification of PrP mRNA expression for knowing the scrapie pathogenesis and providing powerful tool for further studies.