红色原鸡外周血单个核细胞IFN-γ的诱导表达及其荧光定量PCR检测

生物技术通报 ›› 2014, Vol. 0 ›› Issue (4): 102-108.

红色原鸡外周血单个核细胞IFN-γ的诱导表达及其荧光定量PCR检测

孙亭亭¹, 冀斌¹, 马志亮¹, 胡文丽¹, 鲁琼芬¹, 张雄伟², 陈培富¹

(1.云南农业大学动物科学技术学院，昆明 650201；
2. 云南省红河州金平县畜牧局，金平 661500)

收稿日期:2013-12-20 出版日期:2014-04-29 发布日期:2014-04-29
作者简介:孙亭亭，女，硕士研究生，研究方向：兽医微生物学与免疫学；E-mail：sunting915@yeah.net
基金资助:
国家自然科学基金项目(31060130)

Induced Expression and Real-time PCR Detection of IFN-γ from PBMC in Red Jungle Fowl

Sun Tingting¹, Ji Bin¹, Ma Zhiliang¹, Hu Wenli¹, Lu Qiongfen¹, Zhang Xiongwei², Chen Peifu¹,

(1.College of Animal Science and Technology，Yunnan Agricultural University，Yunnan Kunming 650201；
2. Bureau of Animal Production and Health，Yunnan Jinping 661500)

Received:2013-12-20 Published:2014-04-29 Online:2014-04-29

摘要/Abstract

摘要： 为建立检测红色原鸡外周血单个核细胞(PBMC)中干扰素γ(IFN-γ)mRNA表达水平的方法，通过优化刀豆蛋白A(Con A)诱导PBMC表达IFN-γ的条件，采用RT-PCR方法以3-磷酸甘油脱氢酶(GAPDH)基因为内参扩增IFN-γ基因，插入克隆载体pMD18-T分别构建重组质粒pMD-IFN-γ和pMD-GAPDH，以其作为标准品采用SYBR Green I染料法建立荧光定量PCR检测方法，并将该方法应用于34只红色原鸡PBMC IFN-γ表达量的检测。结果发现，PBMC在20 μg/mL Con A刺激培养12 -25 h 时IFN-γ处于高表达水平；标准品质粒pMD-IFN-γ和pMD-GAPDH分别在拷贝数6.72×10²-6.72×10⁹、3.94×10²-3.94×10⁹范围内与其对应的Ct值呈现良好的线性关系(R² > 0.99)，扩增产物的熔解曲线均只有特异的单峰，表明所用引物特异性强。在优化Con A诱导红色原鸡PBMC表达IFN-γ条件的基础上，建立了可用于定量检测红色原鸡PBMC IFN-γ mRNA表达水平的荧光定量PCR方法。

关键词: 红色原鸡, 干扰素γ, Con, A诱导, 荧光定量PCR, 外周血单个核细胞

Abstract: In order to establish the method for detection of interferon-gamma(IFN-γ)mRNA expression in PBMC of red jungle fowl(RJF), the induction conditions of IFN-γ expression were optimized. IFN-γ gene and 3-phosphoric acid glycerol dehydrogenase(GAPDH)gene as an internal control were respectively amplified from the total RNA by RT-PCR and cloned into a pMD18-T vector, to obtain the recombinant plasmids(pMD-IFN-γ and pMD-GAPDH)which served as templates to map the standard curve of real-time PCR with SYBR Green I staining. This method was then applied to 34 RJF. The results showed that IFN-γ was expressed at high levels when PMBC were stimulated with 20 μg/mL of Con A for 12 - 25 h. A good linear correlation(R²>0.99)was observed as the templates ranged from 6.72×10²-6.72×10⁹copies for IFN-γ and 3.94×10²-3.94×10⁹ copies for GAPDH, and the melting curves presented single peaks, respectively. Based on the optimized condition of Con A-induced IFN-γ expression in PBMC, real-time PCR method was successfully established for quantitative analysis of RJF IFN-γ mRNA.

Key words: Red Jungle, Fowl Interferon-γ, Con A induction, Real-time, PCR, PBMC

孙亭亭, 冀斌, 马志亮, 胡文丽, 鲁琼芬, 张雄伟, 陈培富. 红色原鸡外周血单个核细胞IFN-γ的诱导表达及其荧光定量PCR检测[J]. 生物技术通报, 2014, 0(4): 102-108.

Sun Tingting, Ji Bin, Ma Zhiliang, Hu Wenli, Lu Qiongfen, Zhang Xiongwei, Chen Peifu,. Induced Expression and Real-time PCR Detection of IFN-γ from PBMC in Red Jungle Fowl[J]. Biotechnology Bulletin, 2014, 0(4): 102-108.

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