Cloning And Prokaryotic Expression Of Guangxi Swamp Type Buffalo Interferon-γ Gene

Interferon is a kind of highly active and multi-functional glucoprotein produced by activated cells. Interferon is devided into class I and class II. Interferon-γ (IFN-γ) belongs to class II. Buffalo IFN-γ is an antiviral cytokine produced by activated T cells and NK cells. It upregulats MHCII antigen expression, influences the cellular and humoral immune responses and has pleiotropic regulatory effects on immune system.In this work, the total RNA was extracted from peripheral blood mononuclear cells (PBMC), isolated from three healthy swamp type buffalo and stimulated with ConA for 13 and 17 hours, respectively. And the cDNA of buffalo IFN-γ gene was amplified by RT-PCR using a pair of primers designed according to the published bovine IFN-γ gene sequence. The PCR product was cloned into pMD18-T vector. Then, six of the positive recombinant clones were sequenced and analyzed. Comparison of the sequenced IFN-γ with the reported Indian buffalo IFN-γ sequence in GeneBank revealed 99.6% identity at the nucleotide level, and shares 97.8%, 96.0%, 85.8%, 82.4%, 81.0%, 75.0% and 60.7% identities with bovine, goat, pig, dog, horse, human and mouse IFN-γ gene in nucleotide, respectively. Comparing with the reported Indian buffalo IFN-γ sequence, there was 100% identity at the amino acid level, and shares 97.6%, 95.2%, 77.1%, 77.1%, 76.5%, 60.8% and 40.6% identities with bovine, goat, pig, dog, horse, human and mouse IFN-γ gene in amino acid, respectively.The buffalo IFN-γ gene was subcloned into an expression vector pET32a+. Thus, recombinant expression plasmid pET-mIFN-γ was constructed. The...