斑节对虾胸腺素β5的克隆、表达与功能分析  

Cloning, expression and functional analysis of β5 of Penaeus monodon

作　　者：陈鸣 赵超[2] 范嗣刚[2] 王鹏飞[2] 闫路路 王芳 邱丽华 CHEN Ming;ZHAO Chao;FAN Sigang;WANG Pengfei;YAN Lulu;WANG Fang;QIU Lihua(Key Laboratory of Exploration and Utilization of Aquatic Resources,Ministry of Educaction,National Demonstra-tion Center for Experimental Fisheries Science Educection,College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of South China Sea Fishery Resources Exploitation&Utilization,Ministry of Agriculture and Rural Affairs,South China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510300,China;College of Fisheries and Life Science,Dalian Ocean University,Dalian 116023,China)

机构地区：[1]上海海洋大学水产与生命学院,水产科学国家级实验教学示范中心,水产种质资源发掘与利用教育部重点实验室,上海201306 [2]中国水产科学研究院南海水产研究所,农业农村部南海渔业资源开发利用重点实验室,广东广州510300 [3]大连海洋大学水产与生命学院,辽宁大连116023

出　　处：《中国水产科学》2020年第4期383-392,共10页Journal of Fishery Sciences of China

基　　金：国家重点研发计划专项(2018YFD0900303);海南省自然科学基金项目(319QN335);中央级公益性科研院所基本科研业务费专项(2018HY-ZD0204);海南省重点研发计划项目(ZDYF2018163);广东省促进经济发展专项(GDOE[2019]A24).

摘　　要：β-胸腺素(β-thymosin)是一类高度保守的功能多肽,在伤口愈合、血管生成、抗菌和抗病毒免疫等生理活动中起重要的调控作用。本研究从斑节对虾(Penaeus monodon)中成功克隆了一种β-thymosin cDNA序列(β-thymosin 5),其全长为1495 bp,开放阅读框(open reading frame,ORF)为615 bp,编码204个氨基酸。多重序列比对和系统进化树分析结果表明Pmβ5与对虾属的日本囊对虾(Penaeus japonicus)和凡纳滨对虾(Penaeus vannamei)的胸腺素β5聚为一支,其中与日本囊对虾的胸腺素β5同源性最高,相似性高达77%。组织分布研究显示Pmβ5 mRNA在肝胰腺和淋巴等免疫相关组织中的表达量明显高于其他组织。在灭活的副溶血弧菌(Vibrio parahemolyticus)刺激下,Pmβ5基因的表达量显著升高(P<0.05),这说明Pmβ5参与了对虾的抗菌免疫反应。此外,用原核表达技术获得了Pmβ5的体外重组蛋白,抗菌实验(牛津杯法)检测证明,Pmβ5对哈维氏弧菌、溶藻弧菌、副溶血弧菌和枯草芽孢杆菌均有抗菌活性。Penaeus monodon is the largest-culturing species of shrimp,which lends to its high economic and natural nutritional value.However,diseases caused by viruses and bacteria often occur in this species,causing huge economic losses to the shrimp farming industry.Since shrimp can only rely on their innate immune system to protect against pathogen invasions,it is important to understand this innate immune system by studying the immune related molecules of shrimp species,such as P.monodon.This understanding is essential for the prevention and treatment of shrimp dis-eases.It has been reported thatβ-thymosins are a class of highly conserved functional peptides that play important regulatory roles in physiological activities such as wound healing,angiogenesis,and antibacterial and antiviral immuni-ty.In this experiment,oneβ-thymosin gene sequence was successfully cloned from Penaeus monodon.The full-length cDNA was 1495 bp and the open reading frame(ORF)was 615 bp,which encoded 204 amino acids.It was named Pmβ5 because it contained 5 thymosin beta actin-binding motifs(THY).Multiple sequence alignment and phylogenetic tree analysis showed that Pmβ5 was clustered with thymosinβ5 of Penaeus japonicus and Penaeus vannamei,and had the highest homology with thymosinβ5 of Penaeus japonicus,with a similarity of 77%.Real-time quantitative PCR(qRT-PCR)showed that the Pmβ5 gene was expressed in the hepatopancreas,gills,intestine,stomach,muscle and lymphoid organ,and was significantly higher in immune related tissues such as the hepatopancreas and lymphoid organ.The in vitro recombinant protein of Pmβ5 was obtained by a prokaryotic expression technique and the recombinant protein was confirmed to be a Pmβ5 protein by Western Blot analysis.The antibacterial activity of the recombinant protein was further confirmed by the antibacterial experiment(Oxford Cup method).It was found that the recombinant protein had certain antibacterial activity against four bacte-ria.After 12 hours of inactivation by inactivated Vibrio parahemolyti

关 键 词：斑节对虾 β-thymosin 基因表达 原核表达 抗菌活性 

分 类 号：S917[农业科学—水产科学]