Molecular characterization, tissue distribution and functional analysis of macrophage migration inhibitory factor...

The macrophage migration inhibitory factor (MIF) produced in numerous cell types is a multi-functional cytokine mediating both innate and adaptive immune responses. To obtain a better understanding of the innate immune ability of Andrias davidianus and their defensive mechanisms, we identify the A. davidianus MIF (AdMIF) cDNA sequence from a skin cDNA library. The full-length cDNA of AdMIF was of 661bp, consisting of 134bp 5'-terminal UTR, 348bp open reading frame and 179bp 3'-terminal UTR. The deduced protein was composed of 115 amino acids, with an estimated molecular mass of 12.53kDa and a predicted pI of 6.07. AdMIF primary structure is as conserved as the other known sequences. Real time quantitative PCR (qRT-PCR) analysis indicated that AdMIF gene was ubiquitously expressed in selected tissues, with the highest level in liver, moderate level in spleen, intestine, stomach, and the lowest level in heart and skin. The cDNA fragment encoding mature peptide of AdMIF was recombined and expressed in Escherichia coli BL21 (DE3). By means of Ni(2+)-chelating chromatography, the recombinant protein of AdMIF (rAdMIF) was purified successfully. The rAdMIF protein was proved to have enzymatic redox and tautomerase activity in vitro. This study represents the first report for characterization of A. davidianus MIF, demonstrating the successful isolation of MIF from Chinese giant salamanders, and the purified rAdMIF protein is important to produce the monoclonal antibodies and provides a foundation for further investigation of the physiological function of AdMIF.