鰤鱼诺卡氏菌SOD基因克隆与表达及其酶活性质测定  

Gene Cloning, Expression and Enzyme Activity Identification of SOD Gene from Nocardia seriolae

作　　者：侯素莹 黄嘉慧 陈建林 王文基[1,2,4] 鲁义善 夏立群[1,2,3] Hou Suying;Huang Jiahui;Chen Jianlin;Wang Wenji;Lu Yishan;Xia Liqun(Shenzhen Research Institute of Guangdong Ocean University,Shenzhen,518108;Fisheries College of Guangdong Ocean University,Zhanjiang,524088;Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment,Shenzhen,518108;Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals,Zhanjiang,524088)

机构地区：[1]广东海洋大学深圳研究院,深圳518108 [2]广东海洋大学水产学院,湛江524088 [3]广东省水生动物健康评估工程技术研究中心,深圳518108 [4]广东省水产经济动物病原生物学及流行病学重点实验室,湛江524088

出　　处：《基因组学与应用生物学》2019年第11期4894-4901,共8页Genomics and Applied Biology

基　　金：广东省自然科学基金(2017A030313179);深圳市科技计划(JCYJ20170306161613251);深圳大鹏新区产业发展专项(KY20160207);广东海洋大学自然科学研究项目(C17377;C13454);广东省科技发展专项资金项目(2016A050502061);广东省大学生创新创业训练计划(201710566042)共同资助

摘　　要：鱼类的诺卡氏菌病主要由鰤鱼诺卡氏菌(Nocardia seriolae)引起。为探讨鰤鱼诺卡氏菌的毒力因子及其感染致病机制,本研究通过对鰤鱼诺卡氏菌全基因组序列的分析,发现了一个编码超氧化物歧化酶(superoxide dismutase,SOD)的基因。生物信息学分析显示该基因编码一个分泌蛋白,可能是鰤鱼诺卡氏菌潜在的毒力因子。本研究通过基因克隆获取了鰤鱼诺卡氏菌SOD基因(NsSOD),其长度为624 bp。利用双酶切技术在含有GST标签的原核表达载体pGEX-6P-1中插入NsOD基因片段,成功构建了其重组表达载体并命名为pGEX-SOD。将pGEX-SOD导入大肠杆菌BL21(DE3)感受态细胞,获得了原核表达菌株BL21/pGEXSOD并对其诱导表达条件进行了摸索,确定25℃、IPTG(1 mmol/L)诱导14 h,可成功获得具有生物活性的可溶性NsSOD重组蛋白。随后利用GST标签亲和柱纯化NsSOD重组蛋白,并进行酶活性质测定,确定NsSOD重组蛋白的活性为2.76酶活力单位。通过对NsSOD进行基因克隆、原核表达、重组蛋白纯化及体外酶活性质测定,为进一步研究NsSOD的功能和深入了解鰤鱼诺卡氏菌的致病机理奠定了基础。Nocardia seriolae is the main pathogen of fish nocardiosis.To investigate the virulence factors and pathogenic mechanism of N.seriolae,this study found a gene encoding superoxide dismutase(SOD)by analyzing the whole genome sequence of N.seriolae.Bioinformatics analysis showed that the SOD gene of N.seriolae(NsSOD)encoded a secreted protein and may be the virulence factor of N.seriolae.The 624 bp N.seriolae SOD gene(NsSOD)was obtained by gene cloning,and inserted into the prokaryotic expression vector,pGEX-6P-1 with GST tag by Double Enzyme Digestion.The recombinant expression vector was named pGEX-SOD and then transformed to E.coli BL21(DE3)for expression under the induction of IPTG(1 mmol/L)at 25℃for 14 h.The soluble NsSOD recombinant protein with biological activity was successfully expressed.Then the recombinant NsSOD protein was purified by GST agarose resin column and its enzyme activity was identified to be 2.76 units.In this study,the gene cloning,prokaryotic expression,recombinant protein purification and enzyme activity identification of NsSOD laid a foundation for further function study on NsSOD and promoted the understanding of the pathogenic mechanism of N.seriolae.

关 键 词：鰤鱼诺卡氏菌 超氧化物歧化酶 原核表达 蛋白纯化 酶活性质测定 

分 类 号：S943[农业科学—水产养殖]