摘要
为揭示太平洋鳕Gadus macrocephalus抗菌肽Hepcidin(GmHep)的分子结构和表达特性,利用qPCR法对平均体质量为2.438 kg鳕不同组织和发育早期的GmHep表达模式进行了探讨,分别构建了原核(pET-32a-GmHep)和真核(pEGFP-GmHep)表达载体,研究了GmHep的体外表达特点。结果表明:GmHep基因开放阅读框长为297 bp,编码98个氨基酸,蛋白相对分子质量为10833.56,该肽N端具22个氨基酸残基的信号肽,成熟肽含有8个半胱氨酸残基、4个二硫键;氨基酸序列比对发现,GmHep与大西洋鳕Hepcidin同源性最高(93%),但与其他鱼类Hepcidin同源性较低(小于60%),预示其功能具有独特性;GmHep在肝脏中的表达量显著高于其他组织(P<0.05),且GmHep在5、10、30、37、40日龄仔稚鱼中均有表达(P<0.05);GmHep体外诱导表达优化条件为IPTG浓度0.01 mmol/L、30℃、诱导4 h;GmHep在EPC细胞系中主要定位在细胞质中。研究表明,本研究成功构建了两种GmHep体外表达载体,确定GmHep为肝分泌型,且在太平洋鳕发育早期发挥一定作用。
The expression patterns of GmHep were investigated in liver,kidney,spleen,brain,intestine,gill and muscle of Pacific cod Gadus macrocephalus with average body weight of 2.438 kg and in 5,10,30,37 and 40 day old larvae using qPCR,and the prokaryotic(pET-32a-GmHep)and eukaryotic(pEGFP-GmHep)expression vectors were constructed in order to reveal the molecular structure and expression characteristics of the Hepcidin(GmHep),and to develop a new type of antimicrobial peptide.It was found that the GmHep gene had an open reading frame of 297 bp length,encoding 98 amino acids,and molecular weight of 10833.56.There were 22 amino acid residues on the N-terminal of the peptide which mature peptide contained 8 cysteine residues and 4 disulfide bonds.The sequence alignments showed that the Hepcidin homology between Pacific cod and Atlantic cod G.morhua was the maximaum(93%),and the homology of GmHep and other fish was lower(less than 60%),indicating that the function of GmHep was unique.The expression level of GmHep was higher in liver than that in other tissues,and GmHep was expressed in 5,10,30,37,40-day old larvae and juveniles,indicating that GmHep can play roles in the early stage of development.The optimal inducing expression of GmHep in vitro was observed under the conditions of IPTG concentration of 0.01 mmol/L,30℃,and induction for 4 hours.GmHep was found to be mainly located in the cytoplasm of carp epithelioma cell(EPC)line.In conclusion,two kinds of GmHep expression vectors were successfully constructed in vitro,and it was approved that GmHep is a liver secretion type,and it plays a certain role in the early development of Pacific cod.The findings provide a basis for the development of new antimicrobial peptides in fish.
作者
吴玲梅
顾杰
温智清
贾鹏
刘瑞婷
蒋洁兰
刘荭
郑晓聪
毛明光
WU Lingmei;GU Jie;WEN Zhiqing;Jia Peng;LIU Ruiting;JIANG Jielan;LIU Hong;ZHENG Xiaocong;MAO Mingguang(Key Laboratory of Mariculture and Stock Enhancement in North China s Sea,Ministry of Agriculture and Rural Affairs,Key Laboratory of Fish Applied Biology and Aquaculture in North China,Liaoning Province,Dalian Ocean University,Dalian 116023,China;Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center,Shenzhen 518045,China;Shenzhen Academy of Inspection and Quarantine Sciences,Shenzhen 518045,China)
出处
《大连海洋大学学报》
CAS
CSCD
北大核心
2020年第1期83-88,共6页
Journal of Dalian Ocean University
基金
辽宁省高等学校创新人才项目(LR2016009)
辽宁省教育厅科研项目(JL2019021,QL201702)
关键词
太平洋鳕
抗菌肽
原核表达
亚细胞定位
Gadus macrocephalus
Hepcidin
prokaryotic expression
subcellular localization
