日本鳗鲡Ⅰ型Cathelicidin基因的克隆与原核表达  

Cloning and Prokaryotic Expression of Cathelicidin Gene from Japanese Eel Ⅰ,Anguilla japonica

作　　者：张东玲[1] 喻达辉[2] 

机构地区：[1]集美大学水产学院鳗鲡现代产业技术教育部工程研究中心,厦门361021 [2]中国水产科学研究院南海水产研究所,广州510300

出　　处：《生物技术通报》2015年第7期124-131,共8页Biotechnology Bulletin

基　　金：福建省教育厅重点项目(JA13171);集美大学李尚大学科建设基金项目(ZC2013003);集美大学鳗鲡现代产业技术教育部工程研究中心开放基金项目(ZK2013005)

摘　　要：Cathelicidin是目前发现的一个最大抗菌肽家族,具有多重生物学功能。旨在探索和利用鱼类Cathelicidin抗菌机制和潜在的生物学功能。应用RACE-PCR技术获得日本鳗鲡I型Cathelicidin(Aj Cath I)基因的c DNA全长序列。Aj Cath I的c DNA全长为842 bp,开放阅读框为570 bp,编码189个氨基酸。氨基酸序列分析表明,Aj Cath I靠近C端有4个保守的半胱氨酸序列,抗菌成熟肽与香鱼(Plecoglossus altivelis)抗菌成熟肽相似性最高,同源性为65.57%。进化分析表明,Aj Cath I与其它鱼类亲缘关系较近,处于同一个分支上。将Aj Cath I基因克隆至p ET-28a载体,转化Escherichia coli BL21(DE3)宿主菌进行表达。结果表明,Aj Cath I以包涵体形式表达,经鎳柱纯化、Western blot验证和透析复性,获得高纯度的重组蛋白。Cathelicidin, an utmost family of antibacterial peptide so far, possesses multiple biological functions. In order to explore and exploit the antibacterial mechanism and potential biological functions of Cathelicidin, full-length sequence of cDNA of Cathelicidin gene from Japanese eel Anguillajaponica I （ AjCathl ） was obtained by RACE-PCR. The full cl）NA of AjCathl was 842 bp and the ORF contained 570 bp encoding 189 amino acids. Analysis of amino acid sequence demonstrated that AjCathl contained 4 conservative cysteine residues at C terminal, and the mature antibacterial peptide had the highest similarity with Plecoglossus altivelis by homology of 65.57%. Phylogenetic analysis revealed that AjCathl shared a common evolutionary origin with other teleost fishes. AjCathl was cloned into pET-28a and expressed in Escherichia coli BL21 （ DE3 ） . The result manifested that AjCathI protein was expressed in inclusion bodies. The protein was isolated by Ni column, identified by Western blot and re-natured by dialysis, the protein of high purity was gained. This study laid the foundation for further verifying mature peptide sequence of AjCathI and studying its antibacterial activities as well as other biological functions.

关 键 词：日本鳗鲡 CATHELICIDIN 抗菌肽 RACE-PCR 

分 类 号：S917.4[农业科学—水产科学] Q78[生物学—分子生物学]