树鼩 IL- 2 TaqMan 探针实时荧光定量RT-PCR 检测方法的建立
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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

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Establishment of a real-time flurescent quantitative RT-PCRassay for detection of tree threw IL-2 gene
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1. Center of Tree Shrew Germplasm Resources,Institute of Medical Biology,the Chinese Academy of Medical Science and Peking Union Medical College. Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases. Kunming 6501182. Faculty of Life Science and Technology,Kunming University of Science and Technology,Kunming 650500,China

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    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。

    Abstract:

    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 realtime 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.

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黄晓燕,徐 娟,王文广,殷安国,李晓飞,孙晓梅,夏雪山,代解杰.树鼩 IL- 2 TaqMan 探针实时荧光定量RT-PCR 检测方法的建立[J].中国比较医学杂志,2013,23(7):36~40+49.

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  • 收稿日期:2013-03-28
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  • 在线发布日期: 2025-11-07
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