日本鳗鲡巨噬细胞移动抑制因子的基因鉴定及表达分析

巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)是一类进化上古老的多功能细胞因子,广泛分布于细菌、植物和动物中。哺乳动物MIF兼具酶催化活性和趋化作用,在机体炎症反应中具有十分重要的作用。为探究MIF在鱼类免疫系统中的作用,本实验利用PCR技术克隆获得了日本鳗鲡MIF基因(AjMIF)。预测的AjMIF前体肽含MIF特征性的硫醇蛋白氧化还原酶活性基序Cys57-Ala-Leu-Cys60,以及异构酶活性相关的保守氨基酸残基,如Pro2和Cys81等。荧光定量结果显示,AjMIF在日本鳗鲡不同组织中均有表达,且在肝脏中表达量最高,其次为中肾和肠。脂多糖刺激8 h后,头肾、中肾和鳔中AjMIF表达量显著上调;PolyI:C刺激8 h后,鳃、皮肤和肠中AjMIF表达量显著上调。迟缓爱德华氏菌人工感染8 h后,肠和鳃中AjMIF表达量极显著上调;感染16 h后,鳃组织中MIF表达量显著升高;感染24 h后皮肤和鳃中MIF基因表达量显著上调。此外,本研究构建了AjMIF原核表达质粒,在获得重组蛋白的基础上研究了rAjMIF异构酶活性。结果显示,1 nmol重组蛋白在pH 6.2时异构酶活性为2.6 U,而在pH 8.0,酶活性为36.6 U。本研究结果为进一步解析MIF在鱼类免疫系统中的作用奠定了基础。

Characterization and expression analysis of MIF gene from Anguilla japonica

Macrophage migration inhibitory factor (MIF) is evolutionarily ancient and has been found across kingdoms including animals, plants and bacteria. In mammals, MIF has been suggested as chemokine-like cytokine with enzymatic activities, which plays a critical role in inflammatory response against pathogen infections. In this study, we cloned a MIF-like (named after AjMIF) gene for the first time in Japanese eel, Anguilla japonica. The AjMIF precursor processes MIF signature sequence motif, Cys₅₇-Ala-Leu-Cys₆₀, for its thiol-protein oxidoreductase activity, and several conserved residues which are critical for its isomerase activity, such as Pro₂ and Cys₈₁. Expression analysis showed that AjMIF is expressed in various organs / tissues with the highest in liver, followed by middle kidney and intestine. The expression level of AjMIF was significantly increased in head kidney, middle kidney and swim bladder after challenged with lipopolysaccharides at 8 hours post-injection (hpi). Significant increase in AjMIF expression was also observed in gill, skin and intestine at 8 hpi following stimulation with polyinosinic-polycytidylic acid. In addition, significant increase of AjMIF mRNA in gill and intestine was observed at 8 hpi, and in gill at 16 and 24 hpi, and in skin at 24 hpi when infected with Edwardsiella tarda. Furthermore, pH-dependent tautomerase activity of AjMIF has also been found by a recombinant rAjMIF using a prokaryotic expression system. Our results showed that 1 nmol of rAjMIF exhibited 2.6 U of tautomerase activity at pH 6.2, but 36.6 U at pH 8.0. Overall, our study provides a basis for future research aiming at a better understanding the functions of MIF in fish immune system.