College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling, Shaanxi, China.
Dev Comp Immunol. 2013 Mar;39(3):161-8. doi: 10.1016/j.dci.2012.10.012. Epub 2012 Nov 22.
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.
巨噬细胞移动抑制因子（MIF）在多种细胞类型中产生，是一种多功能细胞因子，介导先天和适应性免疫反应。为了更好地了解大鲵的先天免疫能力及其防御机制，我们从皮肤 cDNA 文库中鉴定出大鲵 MIF（AdMIF）cDNA 序列。AdMIF 的全长 cDNA 为 661bp，包含 134bp 的 5'UTR、348bp 的开放阅读框和 179bp 的 3'UTR。推导的蛋白质由 115 个氨基酸组成，估计分子量为 12.53kDa，预测 pI 为 6.07。AdMIF 的一级结构与其他已知序列一样保守。实时定量 PCR（qRT-PCR）分析表明，AdMIF 基因在选定的组织中广泛表达，肝脏中的表达水平最高，脾脏、肠道、胃中的表达水平中等，心脏和皮肤中的表达水平最低。AdMIF 成熟肽编码的 cDNA 片段在大肠杆菌 BL21（DE3）中重组表达。通过 Ni(2+)-螯合层析，成功纯化了 AdMIF 的重组蛋白（rAdMIF）。体外实验证明 rAdMIF 蛋白具有酶促氧化还原和互变异构酶活性。本研究首次报道了大鲵 MIF 的特征，成功地从中国大鲵中分离出 MIF，纯化的 rAdMIF 蛋白对于产生单克隆抗体非常重要，并为进一步研究 AdMIF 的生理功能提供了基础。
