猪IL-2、IL-4、IL-10和IFN-γ的TaqMan实时定量RT-PCR检测体系的建立  

Development of a TaqMan real-time RT-PCR assay for detecting porcine IL-2, IL-4, IL-10, and IFN gamma

作　　者：马玉媛[1] 郭逸[1] 颜奇坡[1] 叶小丽[1] 张启模[1] 郑霖[1] 田克恭[2] 章金刚[1] 

机构地区：[1]军事医学科学院野战输血研究所/国家生物医学分析中心病毒安全检测实验室,北京100850 [2]中国动物疫病预防控制中心,北京100094

出　　处：《中国预防兽医学报》2011年第4期285-288,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家科技支撑计划资助项目(2007BAD86B01-3)

摘　　要：为了解猪体内细胞免疫应答机制,本研究建立猪细胞因子IL-2、IL-4、IL-10和IFN-γ的TaqMan实时定量RT-PCR检测体系。以五指山小型猪cDNA为模板,扩增IL-2、IL-4、IL-10和IFN-γ的基因保守区,构建相应的重组质粒,并将其作为标准品构建标准曲线。结果显示,各标准曲线的相关系数r值均大于0.990,扩增效率为90%～105%;该检测方法的敏感性较高,IL-4检测下限为1copies/μL,IL-2、IL-10及IFN-γ检测下限达为102copies/μL;重复性较好,批内重复试验与批间重复试验变异系数均小于5%。本研究为病毒感染后猪体内免疫应答等研究提供方法。To develope a TaqMan real-time RT-PCR assay for detecting porcine IL-2, IL-4, IL-10, and IFN-γ, the conserved regions of porcine IL-2, IL-4, IL-10, and IFN-γ genes were amplified with Wuzhishan miniature pig cDNA as the template and cloned into pMD18-T to construct the standard recombinant plasmids for establishing calibration curve, respectively. The results showed that the real-time RT-PCR assay was sensitivity and repeatability. The detection limit of IL-4 mRNA was 1 copies/μL, while the detction limit of IL-2, IL-10, and IFN-γ was 102 copies/μL, respectively. The coefficient of variation for both intra-assay and inter-assay was lower than 5%. The evaluation results further demonstrated that the established real-time RT-PCR detection was reliable. This method could be used to evaluate the immune response in pigs.

关 键 词：细胞因子 实时定量RT-PCR 细胞免疫 

分 类 号：S852.4[农业科学—基础兽医学]