绵羊朊病毒受体37kD/67kD LRP/LR实时荧光定量PCR标准质粒和标准曲线的构建  

Construction of standard plasmid and standard curve of Real-time RT-PCR for scrapie agent receptor gene-37kD/67kD LRP/LR

作　　者：乔俊文[1] 苏晓鸥[1] 赵德明[1] 李玉荣[2] 王伊琴[1] 

机构地区：[1]中国农业大学动物医学院/国家动物海绵状脑病实验室,北京100193 [2]河北农业大学动物科技学院,河北保定071001

出　　处：《中国农业大学学报》2009年第4期15-20,共6页Journal of China Agricultural University

基　　金：国家科技支撑计划项目(2006BAD06A14)

摘　　要：为实时相对荧光定量PCR检测绵羊组织中朊病毒受体LRP/LRmRNA表达水平,构建目的基因LRP/LR、内参基因β-actin的标准质粒和标准曲线;根据GeneBank中绵羊37 kD/67 kDLRP/LR（LRP/LR）和β-actin基因编码区保守序列,设计特异引物;提取肠道组织mRNA,经反转录RT-PCR扩增,产物回收纯化后,与pGEM-T-easy载体连接,转化大肠杆菌感受态细胞DH5α;质粒提取后经酶切、PCR和测序鉴定后,获得阳性LRP/LR,β-actin重组质粒;将阳性重组质粒107～102梯度稀释作为模板,然后进行实时荧光定量PCR;系统软件自动生成标准曲线及回归方程。结果显示产物溶解曲线峰值单一,说明无引物二聚体及引物特异性高;LRP/LR和-βactin标准曲线相关系数分别为r2=0.999,r2=0.997,说明线性关系好;重复性试验结果显示所构建的重组质粒10次同条件扩增均Ct值具有较好的重现性,且变异率均小于5%,说明标准曲线重复性好,稳定性高。说明成功构建目的基因LRP/LR,内参基因β-actin标准质粒和标准曲线,为实时荧光定量PCR检测绵羊组织中朊病毒受体mRNA表达水平提供了基础。The aim of the present study was to prepare the recombinant plasmid and standard curve of Real-time RT-PCR for the quantitation of mRNA expression level of scrapie agent receptor-37 kD/67 kD LRP/LR and housekeeping gene ,β-actin in ovine tissues. Pairs of specific primer were designed based on the conserved coding region of LRP/LR and ,β-actin genome from online GeneBank. We synthesized the first strand cDNA by reverse transcription PCR after isolated the RNA from the ovine intestine. The amplification products were ligated into the pGEM-T Easy Vector after recycling and purification, and transformed into competent TOP10 cells eventually. Plasmid DNA was purified and identified by restricted enzyme digestion, PCR amplification and sequencing. The nucleotide sequences were analyzed using DNAMAN software. The positive plasmids enter into the Real-time RT-PCR steps after gradient dilution from 10^7 to 10^2. The standard curve and regressive curve were generated automatically by the DNA Continuous Fluorescence Detection system. 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 for the LRP/LR and ,β-actin genes were 0. 999 and 0. 997, respectively, suggesting the strong linear relationship between the two groups. The results of repeatability experiments showed that the Ct values hold well for the recombinant plasmids with 10 times PCR amplification under the same condition, and the coefficient of variation （C. V） was less than 5% among groups, suggesting the great repeatability and stability of the standard curve. Therefore, the construction of the standard plasmid and standard curve established the foundation of the procedures focusing on the quantitation of scrapie agent receptor mRNA expression in ovine tissues related with the 37 kD/67 kD LRP/LR afterwards.

关 键 词：LRP/LR REAL-TIME PCR 绵羊 重组质粒 标准曲线 

分 类 号：S855.99[农业科学—临床兽医学]