俄罗斯鲟肝脏表达抗菌肽2基因的分子特征及抗菌活性分析  

Molecular Characterization and Antimicrobial Activity Analysis of Liver-Expressed Antimicrobial Peptide 2(LEAP-2)Gene in Russian Sturgeon(Acipenser gueldenstaedti)

作　　者：徐倩 王宁宁[2] 白莉 史鲲鹏 沙珍霞 XU Qian;WANG Ningning;BAI Li;SHI Kunpeng;SHA Zhenxia(Aquatic Biotechnology Research Institute,College of Life Sciences,Qingdao University,Qingdao 266071,China;Basic Medicine College,Qingdao University,Qingdao 266071,China;College of Fisheries and Life Science,Dalian Ocean University,Dalian 1116023,China)

机构地区：[1]青岛大学生命科学学院水生生物技术研究院,山东青岛266071 [2]青岛大学基础医学院,山东青岛266071 [3]大连海洋大学水产与生命学院,辽宁大连116023

出　　处：《渔业科学进展》2023年第3期37-51,共15页Progress in Fishery Sciences

基　　金：中国水产科学研究院基本科研业务费(2014A03XK01)资助。

摘　　要：肝脏表达抗菌肽2(liver-expressed antimicrobial peptide 2,LEAP-2)在鱼类先天免疫系统中发挥重要作用。本研究通过RACE技术获得了俄罗斯鲟LEAP-2(Acipenser gueldenstaedti LEAP-2,AgLEAP-2)的全长c DNA序列,对其序列特征和表达水平进行了分析;构建Ag LEAP-2原核表达载体并对重组蛋白进行纯化;进一步采用琼脂稀释法初步检测Ag LEAP-2蛋白的抑菌活性。结果显示,AgLEAP-2基因c DNA全长为622 bp,开放阅读框(ORF)为246 bp,预测编码81个氨基酸,分子量约为11.2 kDa,5′端非编码区为184 bp,3′端非编码区为192 bp。Ag LEAP-2包含一个信号肽(1~25 aa)和一个成熟肽(26~81 aa),成熟肽含有4个保守的半胱氨酸残基,在Cys58-Cys69和Cys64-Cys74的相对位置之间各形成一个二硫键的核心结构。实时荧光定量PCR(qRT-PCR)结果显示,AgLEAP-2在所有健康组织中广泛表达,在肝脏中表达水平最高,在肠道和肌肉中表达水平次之,在鳃中表达量最低。与0 h相比,Ag LEAP-2在感染嗜水气单胞菌(Aeromonas hydrophila)后的不同时间点的肝脏、脾脏、肠道、头肾、鳃和血液组织中均显著上调。此外,重组Ag LEAP-2蛋白(r Ag LEAP-2)在体外对革兰氏阳性菌和革兰氏阴性菌都具有抗菌活性并存在剂量效应。本研究表明,AgLEAP-2可能在俄罗斯鲟的先天免疫系统中发挥重要作用且对多种病原菌有一定的抗菌效果,本研究为开发新的抗菌制剂提供了依据。Antimicrobial peptides(AMPs)are an important part of the innate immune defense system;they can effectively kill viruses,fungi,and bacteria,preventing infection(and even sepsis).AMPs also have other functional roles in immune regulation,anti-tumor activity,angiogenesis,and wound healing.Liver-expressed antimicrobial peptide 2(LEAP-2)is an AMP that has been extensively studied in mammals,birds,and fishes.The earliest fish LEAP-2 study reported the cloning of LEAP-2A and LEAP-2B of rainbow trout.So far,LEAP-2 has been studied in various fishes,including blunt snout bream,grass carp,golden pompano,ayu,and large yellow croaker.These studies found that LEAP-2 generally contains four highly conserved cysteine residues and two disulfide bonds;it can disrupt the structural integrity of bacterial cell membrane,revealing its antibacterial activity with an important role in the innate immune system of fishes.Russian sturgeon(Acipenser gueldenstaedti)is cultivated on a certain scale in China,and Aeromonas hydrophila is its main pathogen.Hemorrhagic ascites and tissue hemorrhage appear after infection,causing huge economic losses to Russian sturgeon aquaculture.As an important innate immunity component,LEAP-2 research is of great significance.At present,studies on AgLEAP-2 have not been reported.To study the molecular characteristics and the transcriptional expression patterns of AgLEAP-2,as well as its antibacterial activity in vitro,the full-length AgLEAP-2 cDNA sequence was obtained by RACE.AgLEAP-2 was cloned and its sequence characteristics were analyzed by bioinformatics.The qRT-PCR method was used to detect AgLEAP-2 in 13 different tissues(liver,intestine,spleen,head kidney,blood,gill,skin,ovary,brain,heart,stomach,metanephros,and muscle)of healthy Russian sturgeon,and the AgLEAP-2 transcriptional expression pattern in immune tissues after infection with A.hydrophila.The AgLEAP-2 prokaryotic expression vector was constructed and the recombinant AgLEAP-2 protein(designated rAgLEAP-2)was purified.The antibacterial activit

关 键 词：肝脏表达抗菌肽2(LEAP-2) 基因表达 抗菌活性 细菌感染 俄罗斯鲟 

分 类 号：S917.4[农业科学—水产科学]