| 摘要: |
| 铁蛋白（Ferritin，Fer）是一类广泛存在于生物体内、并与铁代谢密切相关的铁储存蛋白。本研究通过香鱼（Plecoglossus altivelis）单核/巨噬细胞转录组测序获得铁蛋白亚基基因（PaFer） cDNA全序列，结果显示，PaFer由943个核苷酸组成，开放阅读框为531bp，编码176个氨基酸，预测相对分子质量为20.4kDa，等电点为5.46。氨基酸序列多重比对结果显示，PaFer具有保守的铁蛋白样双铁结构域，与虹鳟（Oncorhynchus mykiss）和大西洋鲑（Salmo salar） Fer M亚基（Fer-M）同源性最高，均为92%；系统进化树分析表明，PaFer与胡瓜鱼、大西洋鲑和虹鳟的Fer-M聚为一个小簇，与虹鳟Fer-M进化相关性最高。实时荧光定量PCR（quantitative real-time PCR，qPCR）结果表明，PaFer mRNA主要在香鱼脾脏中表达量最高，肾脏、鳃、脑和心脏中次之；鳗弧菌（Vibrio anguillarum）感染后，香鱼肝脏、脾脏和肾脏等组织中PaFer mRNA表达显著上调。原核表达了PaFer重组蛋白（rPaFer），rPaFer以包涵体形式表达，镍柱亲和层析纯化重组蛋白，尿素梯度透析法复性rPaFer。铁螯合实验结果显示，当rPaFer浓度为4μg/mL时，螯合铁的能力最大，为43.96%。抑菌实验结果表明，rPaFer对鳗弧菌、溶藻弧菌（Vibrio alginolyticus）和创伤弧菌（Vibrio vulnificus）的最小抑菌浓度分别为1.5625、25.0和100.0μg/mL。综上，PaFer与香鱼抗病原感染的免疫反应紧密相关，可能通过调控铁代谢参与鱼类抵抗微生物的宿主自身免疫过程。本研究为进一步研究鱼类Fer的功能及在病原菌感染免疫中的分子机制奠定基础。 |
| 关键词: 铁蛋白 香鱼 表达 铁结合能力 抑菌作用 |
| DOI：10.11693/hyhz20161000232 |
| 分类号: |
| 基金项目:31372555号，31402333号；浙江省自然科学基金项目，LZ13C190001号，LY17C190003号；宁波大学学科项目，XKl15D238号。 |
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| MOLECULAR IDENTIFICATION, EXPRESSION AND FUNCTION ANALYSIS ON FERRITIN IN AYU PLECOGLOSSUS ALTIVELIS |
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CHEN Meng-Dan, ZHU Kai, XU Zhu-Jie, LI Chang-Hong, MIAO Liang, CHEN Jiong
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Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China
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| Abstract: |
| Ferritin (Fer), a type of iron storage protein, exists in organism in close relationship with iron metabolism. We obtained the cDNA sequence of ferritin gene from ayu (Plecoglossus altivelis), known as Fer gene (PaFer), with de novo transcriptome sequencing of ayu monocytes/macrophages. The full-length of cDNA sequence of this gene was 943 nucleotides, containing a large open reading frame encoding 176 amino acids in a predicted molecular mass of 20.4kDa and a theoretical isoelectric point of 5.46. The multiple sequence alignment of complete amino acid sequences indicated that PaFer featured a conserved ferritin-like diiron domain. PaFer shared the highest amino acid sequence identity (92.0%) with those of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) Fer-M. Phylogenetic tree analysis also confirmed that PaFer, Fer-M of Atlantic salmon and rainbow trout grouped into a small cluster, and PaFer was most closely related to that of rainbow trout. Quantitative real-time PCR (qPCR) analysis showed that PaFer mRNA had the highest expression level in spleen of healthy ayu. After Vibrio anguillarum infection, PaFer transcripts upregulated significantly in the liver, spleen, and kidney. The PaFer was overexpressed in the form of inclusion bodies and a Ni2+ nitrilotriacetic acid (NTA) Sefinose column was used to purify PaFer recombinant protein (rPaFer). Urea gradient dialysis method was used to obtain the refolding protein. Result of iron chelation experiment show that at rPaFer concentration of 4μg/mL, the iron chelation of rPaFer reached peak level (43.96%). In addition, result of antimicrobial activity experiment show that the minimum inhibitory concentration (MIC) of rPaFer against V. anguillarum, V. alginolyticus, and V. vulnificus was 1.5625, 25.0, and 100.0μg/mL, respectively. Therefore, we believe that the PaFer can play an important role in immune response against pathogen infection via regulation of iron metabolism. This understanding may provide a theoretical basis for studying the functions of fish Fer and its mechanisms of regulating fish immune response to pathogens. |
| Key words: ferritin Plecoglossus altivelis expression iron chelation capacity antimicrobial activity |
