Biotechnology Bulletin ›› 2014, Vol. 0 ›› Issue (4): 102-108.

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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. (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 Online:2014-04-29 Published:2014-04-29

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 6.72×102-6.72×109 copies 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