实时荧光定量PCR检测PrP基因表达标准品质粒和标准曲线的构建  

Construction of Recombinant Plasmid and Standard Curve for Detection of PrP Gene Expression by Real-time Quantitative PCR

作　　者：宁章勇[1] 赵德明[1] 杨建民[1] 崔亚利[1] 孟丽平[1] 吴长德[1] 秦秀慧[1] 马李颖[1] 

机构地区：[1]中国农业大学国家动物传染性海绵状脑病实验室,北京100094

出　　处：《中国兽医学报》2005年第6期611-613,共3页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30371062);教育部博士点基金资助项目(20020019006)

摘　　要：对金黄地鼠PrP基因序列进行分析,选择其高度保守区域设计引物,对金黄地鼠各组织提取mRNA,经RTPCR扩增,产物纯化后与pGEM-T-easy连接,转化大肠杆菌DH5α,筛选后得到重组标准品质粒,对重组标准品质粒进行酶切、PCR和测序鉴定,表明PrP基因保守区段已经成功克隆.将107～102拷贝/反应的重组标准品质粒进行荧光定量PCR,10次重复Ct值平均分别为17.50±0.5、21.38±0.2、25.29±0.7、29.12±0.7、31.34±0.4、33.89±0.1,系统自动分析软件显示Ct值与标准品浓度的对数之间存在良好的线性关系,回归系数为0.998.对动力学曲线分析表明,在该反应体系和反应条件下,该标准曲线的灵敏度为102拷贝/反应.荧光PCR PrP基因表达检测标准品质粒和标准曲线的建立,为检测PrP基因的组织特异性表达和动物传染性海绵状脑病发生的分子机理奠定了基础.In order to analyze the PrP gene sequence of golden hamster, the primer expression software was used to obtain a couple of primer from highly conservative region. The mRNA of tissues of golden hamster was extracted and the aim gene segment amplified by RT-PCR. The purified PCR product was connected with pGEM-T-easy vector and then transferred into E. coli DH5α. The standard recombinant plasmid was gained from positive bacterium clone. The enzyme cutting, plasmid PCR and plasmid sequence mensuration showed that the conservative segment of PrP gene was successfully cloned. The Ct value were 17. 50±0. 5,21.38±0.2,25.29±0. 7,29. 12±0. 7.31.34±0. 4 and 33.89±0. 1 respectively with 10^7-10^2 eopies/ μL standard plasmid DNA specimen after real-time quantitative PCR amplification for ten times repeat, which indicate that there is a good linear function in statistics between the Ct value and tbe concentration gradient of standard plasmid DNA specimen. The regression value was 0. 998. Analysis of the dynamic curve, under this condition of the reaction, the sensitive degree is 10^2 copies/μL . The construction of recombinant plasmid and standard curve for real-time quantitative PCR provides the basis for detection of tissue-specific PrP gene expression and investigation of molecular mechanism of animal transmissible spongiform encephalopathies.

关 键 词：实时荧光定量PCR PrP基因 标准曲线 重组质粒 动物传染性海绵状脑病 

分 类 号：S855.99[农业科学—临床兽医学] Q78[农业科学—兽医学]