猪Th1/Th2型细胞因子重组质粒实时荧光定量PCR标准曲线的建立  

Establishment of Standard Curves for Detection of Porcine Th1/Th2 Cytokines Genes Recombinant Plasmids Using Real-time PCR

作　　者：温立斌[1] 何孔旺[1] 杨汉春[2] 郭容利[1] 李成仁[1] 卢维彩[1] 贾付从[1] 

机构地区：[1]江苏省农业科学院兽医研究所,江苏南京210014 [2]中国农业大学动物医学院,北京100094

出　　处：《华北农学报》2009年第B08期5-8,共4页Acta Agriculturae Boreali-Sinica

基　　金：国家"973"计划前期研究专项基金(2007CB116308);江苏省自然科学基金(BK2008351)

摘　　要：依据GenBank登录的猪Th1型细胞因子(IL-2、IL-12、IFN-γ)、Th2型细胞因子(IL-4、IL-6、IL-10)的核苷酸序列设计特异性引物,经RT-PCR技术从猪外周血单个核细胞中扩增和克隆了上述因子的101 bp核苷酸片段。测序鉴定后,将重组质粒10倍系列稀释后作为标准模板,通过实时荧光定量PCR方法,建立了它们各自的标准曲线及直线回归方程。结果表明,该方法具有线性关系好,特异性、敏感性、重复性高等特点,为猪体内IL-2、IL-12I、FN-γ、IL-4、IL-6和IL-10的mRNA水平的定量检测,提供了必要的技术手段。The primers were designed and synthesized based on the nucleotide sequences of porcine Th1/Th2 cytokines available in GenBank. The 101 bp fragments were amplified by RT-PCR from the porcine peripheral blood mononuclear cells（ PBMC）, and then the fragments were cloned and sequenced. The recombinant plasmids were diluted by 10-fold serial and used as the real-time PCR standard templates. Their standard curves and the corresponding linear re- gression equations were obtained. The results indicated that the standard curves were shown to be of high linearity, speci- ficity, sensitivity and reproducibility, and they provided powerful tools for quantification of IL-2, IL-12, IFN-γ, IL-4, IL-6 and IL-10 mRNA in pigs.

关 键 词：TH1/TH2型细胞因子 外周血单个核细胞 重组质粒 实时荧光定量PCR 

分 类 号：S852.42[农业科学—基础兽医学]