树鼩IL-2 TaqMan探针实时荧光定量RT-PCR检测方法的建立  

Establishment of a real-time flurescent quantitative RT-PCR assay for detection of tree threw IL-2 gene

作　　者：黄晓燕[1] 徐娟[1] 王文广[1] 殷安国[2] 李晓飞[1] 孙晓梅[1] 夏雪山[2] 代解杰[1] 

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所,云南省重大传染病疫苗研发重点实验室,树鼩种子资源中心,云南昆明650118 [2]昆明理工大学生命科学与技术学院,云南昆明650500

出　　处：《中国比较医学杂志》2013年第7期36-40,49,共6页Chinese Journal of Comparative Medicine

基　　金：国家科技支撑计划项目(编号:2009BAI83B02-21;2011BAI15B01-21;2012BAI39B01);云南省科技计划重点项目(编号:2006PT07-2);云南省应用基础面上项目(编号:2011FZ211)

摘　　要：目的建立检测树鼩IL-2基因TaqMan探针实时荧光定量PCR方法。方法以经ConA诱导培养的树鼩淋巴细胞总RNA为材料,设计特异性引物,通过RT-PCR技术克隆出树鼩IL-2基因,以构建的树鼩IL-2基因质粒为标准品,建立标准曲线,并进行灵敏度检测。结果建立了树鼩IL-2基因mRNA表达实时荧光定量PCR检测方法,此法检测灵敏度高,线性范围可达102～109拷贝/ul。结论成功建立了树鼩IL-2实时荧光定量PCR方法,此方法灵敏度高、特异性强,检测周期短,为探讨IL-2作用的分子作用机制奠定基础。Objective To establish a real-time fluorescent quantitative RT-PCR assay to detect tree threw （tupaiabelangeri） IL-2. Methods Total RNA was isolated from Con A-stimulated tree shrews＇ spleen lymphocytes. The conservative encoding sequence of interleukin-2 （IL-2） was amplified by RT-PCR, and successfully cloned into pMD-19T vector. The established IL-2 gene vector was used as standard substance and the standard curves were established for the sensitivity evaluation. Results A real-time fluorescence quantitative method of fast, sensitive and reliable detection system was set up. The sensitivity of this method for creating IL-2 gene of tree shrew was 102 ~ 109copies. Conclusions A real- time fluorescence quantitative method of tree shrew IL-2 was successfully established. This sensitive method can serve as a molecular foundation for the study of IL-2 and its role in many clinical diseases.

关 键 词：树鼩 IL-2 实时荧光定量 PCR 

分 类 号：R-332[医药卫生]