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101bp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。
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
Abstract: The primers were designed and synthesized based on the nucleotide sequences of porcine Th1/Th2 cytokinesavailable in GenBank .The 101 bp fragments were amplified by RT -PCR from the porcine peripheral bloodmononuclear 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 linearregression equations were obtained .The results indicated that the standard curves were shown to be of high linearity ,specificity,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.
Key words:
Thl/Th2cytokines,
PBMC,
Recombinantplasmi
中图分类号:
WENLi-bin, HEKong-wang, YANGHan-chun, GUORong-li, LICheng-ren, LUWei-cai, JIAFu-cong. Establishment of Standard Curves for Detection of PorcineThl/Th2 Cytokines Genes Recombinant Plasmids UsingReal-timePCR[J]. ACTA AGRICULTURAE BOREALI-SINICA, doi: 10.7668/hbnxb.2009.S1.002.