褐鳟LEAP-2成熟肽在大肠杆菌中的融合表达  

Fusion expression of brown trout LEAP-2 mature peptide in Escherichia coli

作　　者：刘晨斌 徐革锋[2] 黄天晴 谷伟[2] 陈春生 程琳 史秀兰 王炳谦[2] LIU Chenbin;XU Gefeng;HUANG Tianqing;GU Wei;CHEN Chunsheng;CHENG Lin;SHI Xiulan;WANG Bingqian(School of Life Science and Technology,Harbin Normal University,Harbin 150025,China;Heilongjiang River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Harbin 150070,China;Beijng Aquatic Wildlife Rescue and Conservation Center,Beijing 102100,China)

机构地区：[1]哈尔滨师范大学生命科学与技术学院,哈尔滨150025 [2]中国水产科学研究院黑龙江水产研究所,哈尔滨150070 [3]北京市水生野生动植物救护中心,北京102100

出　　处：《东北农业大学学报》2020年第4期70-77,共8页Journal of Northeast Agricultural University

基　　金：中央级科研院所基本科研业务费专项(2020TD32);现代农业产业技术体系专项资金资助(CARS-46);北京市鲟鱼鲑鳟鱼创新团队项目(BAIC08-2020)。

摘　　要：开发抗菌肽作为饲料添加剂,可避免耐药性产生和药物残留等问题,对褐鳟养殖业健康绿色发展具有重要意义。通过原核表达途径获得褐鳟LEAP-2成熟肽融合表达蛋白,利用同源克隆方法,从褐鳟肝脏组织中克隆得到LEAP-2基因ORF序列,分析其氨基酸序列和蛋白质结构,与其他物种比对并作同源性分析,构建重组表达质粒pET32a-mLEAP-2,原核表达。结果表明,褐鳟LEAP-2基因ORF全长291 bp,编码96个氨基酸,其中氨基端信号肽由27个残基组成,成熟肽含有41个残基;根据比对结果发现,在羧基端存在多个保守氨基酸残基,其中包括4个高度保守半胱氨酸残基,4个半胱氨酸残基之间可形成两对二硫键,推测其与LEAP-2抗菌活性有关;同源性分析显示褐鳟LEAP-2在进化上相对保守,氨基酸序列与大西洋鲑、虹鳟(LEAP-2A)、红点鲑同源性均在94%以上;SDS-PAGE电泳分析显示得到的重组蛋白分子质量约为22.61 ku,与预期一致。研究结果有利于进一步探究褐鳟LEAP-2基因功能,为抗菌肽在养殖上的应用提供理论基础。Research and development of antimicrobial peptides as feed additives can avoid drug resistance and drug residues,which is of great significance to the healthy and green development of brown trout breeding industry.In order to obtain the brown trout LEAP-2 mature peptide fusion expression protein through the prokaryotic expression pathway,the homologous cloning method was used to clone the LEAP-2 gene ORF sequence from brown trout liver tissue,and its amino acid sequence and protein structure were analyzed.By alignment and homology analysis,the recombinant expression plasmid p ET32 a-mLEAP-2 was constructed for prokaryotic expression.The results showed that the brown trout LEAP-2 gene ORF was 291 bp in length and encoded 96 amino acids,of which the amino-terminal signal peptide consisted of 27 residues and the mature peptide contained 41 residues;according to the comparison results,it was found that the multiple conserved amino acid residuesincludedfour highly conserved cysteine residues.Two pairs of disulfide bonds could be formed between these four cysteine residues,which was speculated to be related to the antibacterial activity of LEAP-2;homology analysis showed that brown trout LEAP-2 was relatively conservative in evolution,and its amino acid sequence was more than 94%homologous with Atlantic salmon,rainbow trout(LEAP-2 A)and trout salmon;SDS-PAGE electrophoresis analysis showed that the size of the recombinant protein was approximately 22.61 ku as expected.The research results were beneficial to further explore the function of the brown trout LEAP-2 gene,and provided a theoretical basis for the application of corresponding antibacterial peptides in breeding.

关 键 词：褐鳟 LEAP-2 基因克隆 原核表达 

分 类 号：Q785[生物学—分子生物学]