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

Abstract

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

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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