实时荧光定量PCR构建朊病相关细胞因子标准品质粒和标准曲线  

Construction of Standard Plasmid and Standard Curve of Real-time PCR for Cytokines Related to Prion Disease

作　　者：刘爱玲[1,2] 周向梅[2] 杨鸣琦[1] 刘爱林[3] 赵德明[2] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨陵712100 [2]中国农业大学国家海绵状脑病实验室,北京100193 [3]河北省邯郸市动物疫病预防控制中心,河北邯郸056005

出　　处：《动物医学进展》2010年第6期11-14,共4页Progress In Veterinary Medicine

基　　金：国家科技支撑计划项目(2008BAI54B06)

摘　　要：为能够用实时荧光定量PCR检测PrP106-126作用的鼠巨噬细胞细胞因子的表达水平,构建目的基因IL-1β、TNF-α、IL-6和内参基因β-actin的标准品质粒和标准曲线。根据GenBank中鼠基因编码区保守序列,设计特异性引物;细胞经PrP106-126作用后,提取总RNA。经反转录、PCR扩增、纯化、连接和转化后,提取质粒并测序鉴定,获得IL-1β、TNFα-、IL-6和β-actin质粒;将质粒梯度稀释,进行荧光定量PCR,获得标准曲线及回归方程。结果显示,产物溶解曲线峰值单一,引物特异性高;标准曲线相关系数r2=0.999,表明线性关系好,成功构建了目的基因和内参基因的标准品质粒和标准曲线。The aim of the present study was to prepare the plasmid and standard curve of real-time PCR for the quantitation of mRNA expression level of the inflammatory cytokines, IL-1β,TNF-α,IL-6 and house-keeping gene β-actin induced by Prp106-126 in mouse macrophages. Pairs of specific primer were designed based on the conserved coding region from GenBank. The first strand cDNA was synthesized by reverse transcription PCR after isolated RNA from Ana-lcells was treated by prp106-126. Plasmid DNA was puri- fied and identified by PCR amplification and sequencing after recycling and purification, and transformation. The positive plasmids entered into the RT-PCR steps after gradient dilution. The standard curve and regressive curve were generated automatically. The results showed that no specific amplicons was found according to the derived melting curve of the RT-PCR products, indicating the specification of the present primers. The correlation coefficients of the standard were 0. 999, suggesting the strong linear relationship between the groups. Therefore, the construction of the standard plasmid and standard curve were established.

关 键 词：PrP106-126 REAL-TIME PCR 质粒 标准曲线 

分 类 号：S852.659.27[农业科学—基础兽医学]