| 摘要: |
| 摘要Piscidin类抗菌肽具有广谱的抗菌活性，在鱼类先天性免疫中扮演重要角色。本文对大弹涂鱼（Boleophthalmus pectinirostris）单核/巨噬细胞（monocytes/macrophages，MO/MΦ）转录组测序获得piscidin 1基因（Bppis1） cDNA全序列。Bppis1基因cDNA序列由327个核苷酸组成，开放阅读框为207bp，编码68个氨基酸，预测相对分子量为7.7kDa，等电点为5.51。氨基酸序列多重比对分析表明，Bppis1具有piscidin家族的特征结构，信号肽序列最为保守，终止于GEG序列后，与黄条（鱼师）（Seriola lalandi） piscidin同源性最高，为40.8%；系统进化树分析表明，Bppis1属于piscidin 1类，与黄条（鱼师） piscidin进化相关性最高。实时荧光定量PCR （quantitative real-time PCR，RT-qPCR）结果显示，Bppis1 mRNA在健康鱼鳃中表达量最高；迟缓爱德华氏菌（Edwardsiella tarda）感染后，大弹涂鱼肝、脾、肾、鳃和皮肤中Bppis1 mRNA表达量显著上调。体外抑菌实验结果表明，人工合成的Bppis1成熟肽抑菌活性较广泛，但对迟缓爱德华氏菌和3株革兰氏阳性菌无抑菌活性。大弹涂鱼MO/MΦ经1.0μg/mL Bppis1成熟肽处理后，对FITC标记的创伤弧菌（Vibrio vulnificus）的吞噬活性显著增加。综上，Bppis1在大弹涂鱼先天免疫中发挥重要作用，可能作为抵抗病原体入侵的潜在治疗药物。 |
| 关键词: 大弹涂鱼 Piscidin 迟缓爱德华氏菌 抗菌活性 吞噬活性 |
| DOI：10.11693/hyhz20171100297 |
| 分类号:Q786;S917 |
| 基金项目:国家自然科学基金项目，31402333号；浙江省自然科学基金项目，LY14C030002号；宁波市科技创新团队项目，2015C110018号；宁波市自然科学基金项目，2017A610284号，2017A610285号；浙江省生物工程重中之重学科项目，ZS2017005号。 |
附件 |
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| PISCIDIN 1 OF MUDSKIPPER BOLEOPHTHALMUS PECTINIROSTRIS: MOLECULAR CHARACTERIZATION AND FUNCTIONAL ANALYSIS |
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GUAN Feng1,2, LI Chang-Hong1, NIE Li1, MIAO Liang1, CHEN Jiong1
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1.School of Marine Sciences, Ningbo University, Ningbo 315211, China;2.Zhejiang Wanli University, Ningbo 315100, China
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| Abstract: |
| Piscidin-like antimicrobial peptides, which have broad-spectrum activities, exert an important role in fish's innate immunity. In this study, we obtained the complete cDNA sequence of piscidin 1 (Bppis1) from mudskipper Boleophthalmus pectinirostris, with de novo transcriptome sequencing of mudskipper monocytes/macrophages (MO/MΦ). The full-length of cDNA sequence of Bppis1 was 327 nucleotides, containing a 207 nucleotides open reading frame (ORF) encoding 68 amino acids. The deduced molecular weight and isoelectric point of the deduced protein was 7.7kDa and 5.51 respectively. The multiple sequence alignment of complete amino acid sequences revealed that Bppis1 possessed the piscidin family signature motifs, and a conserved sequence in signal peptide terminating at the motif GEG. Bppis1 shared the highest amino acid sequence identity (40.8%) with that of yellowtail amberjack (Seriola lalandi). In addition, the phylogenetic tree confirmed that Bppis1 belonged to fish piscidin 1 cluster and was most closely related to yellowtail amberjack piscidin. Quantitative real-time PCR (RT-qPCR) analysis showed that Bppis1 mRNA had the highest expression level in gill of healthy mudskipper. Upon infection with Edwardsiella tarda, Bppis1 mRNA expression increased significantly in the liver, spleen, kidney, gill and skin. The result of antimicrobial test showed that the synthetic mature peptide of Bppis1 exhibited relatively broad antimicrobial activity in vitro, but no antimicrobial activity against Edwardsiella tarda and three gram-positive bacteria. After treatment with 1.0μg/mL Bppis1 mature peptide, the phagocytosis of FITC-Vibrio vulnificus by MO/MФ was enhanced significantly. Therefore, Bppis1 plays a critical role in the innate immune system of mudskipper, and may be a potential therapeutic agent against pathogen invasion. |
| Key words: Boleophthalmus pectinirostris piscidin Edwardsiella tarda antimicrobial activity phagocytic activity |
