三疣梭子蟹C-型溶菌酶基因的原核表达与活性检测  

Expression of Portunus trituberculatus Lysozyme Gene in E.coli and Evaluation of Its Lytic Activity

作　　者：张辉[1] 潘鲁青[1] 岳峰[1] 

机构地区：[1]中国海洋大学海水养殖教育部重点实验室,山东青岛266003

出　　处：《中国海洋大学学报（自然科学版）》2013年第7期23-27,共5页Periodical of Ocean University of China

基　　金：国家高技术研究发展计划项目(2012AA10A409)资助

摘　　要：溶菌酶能破坏和消除侵入体内的病原或异物,是甲壳动物免疫系统中一个重要的效应分子。本文根据克隆得到的三疣梭子蟹(Portunus trituberculatus)C-型溶菌酶基因(Lysozyme)全长序列,设计带有酶切位点的特异性引物,经RT-PCR扩增得到溶菌酶基因(PtLys)的开放性阅读框,将其克隆至载体pMD18-T,并测序。用限制性内切酶BamH I和XhoI双酶切取目的基因,并与表达载体pET-28a(+)连接,构建了PtLys基因的原核表达载体p28a-PtLys,在大肠杆菌(E.co-li)BL21(DE3)中诱导表达含6个His标签的重组PtLys融合蛋白,经带有His标签的Ni 2+亲合层析柱纯化得到三疣梭子蟹重组溶菌酶蛋白,并进行活性检测。结果表明:重组载体p28a-PtLys转化E.coli BL21(DE3)后,可以实现PtLys基因的原核表达,经SDS-PAGE分析显示在33.29kD处有显著诱导条带,与预测的分子量大小基本一致;经Western blot分析,可与anti-His抗体特异性结合;抑菌实验表明,对溶藻弧菌(Vibrio alginolyticus)、哈维氏弧菌(Vibrio harveyi)和鳗弧菌(Vibrio anguillarum)均有一定的抑菌作用。本研究成功实现了三疣梭子蟹C-型溶菌酶基因的原核表达,为甲壳动物溶菌酶的进一步开发应用提供了技术支持。Lysozyme is an important responsive molecule in the immune system of crustacean against the invasion of bacterial pathogens or foreign stuff. The total RNA extracted from the hemocyte sample of Portunus trituberculatus was used to amplify the DNA sequence encoding an open reading frame of lysozyme gone （named PtLys） with RT-PCR. The sequence was then inserted into pMD18-T vector and sequenced. The recombinant plasmid was digested by Barn H I and Xho I, with the gene inserted into pET-28a （-F） digested with the same pair of enzymes. The recombinant p28a-PtLys was transformed into E. coli BL21 （DE3） which was induced by isopropylthio-13-D-galactoside （IPTG）. The recombinant protein was purified through His-Band resin chelating chromatography, and [ytic activity was assayed finally. In conclusion, PtLys in p28a-PtLys expressed successfully in E. coli BL21（DE3）. SDS-PAGE revealed that the molecular weight of PtLys was 33.29KD, being consistent with the predicted. Western blotting revealed that the recombinant protein bound with anti-His body specifically. PtLys displayed antibacterial activity against Vibrio alginolyticus, V. harveyi and V. anguilIarum. This study successfully expressed lysozyme of Portunus trituberculatus in E. coli, which provided a technical support for the application of lysozyme in crustaceans.

关 键 词：三疣梭子蟹 C-型溶菌酶 原核表达 抑菌活性 

分 类 号：X835[环境科学与工程—环境工程]