翘嘴鳜铜锌超氧化物歧化酶基因的分子克隆与原核表达  

Cloning and prokaryotic expression of copper/zinc superoxide dismutase gene fromSiniperca chuatsi

作　　者：程周玉[1] 许亮清 胡向萍[1] 胡宝庆[1] 简少卿[1] 阳刚[1] 张明[3] 文春根[1] 宁瑞红 王鹤[1] 

机构地区：[1]南昌大学生命科学学院,江西南昌330031 [2]南昌市农科院畜牧水产研究所,江西南昌330038 [3]江西生物科技职业学院,江西南昌330200

出　　处：《南昌大学学报（理科版）》2015年第6期587-596,共10页Journal of Nanchang University(Natural Science)

基　　金：南昌市科技支撑计划基金资助项目(2012-KJ2C-NY-SC-002);国家自然科学基金资助项目(31472305,31460697,21467015);江西省教育厅基金资助项目(GJJ12024,GJJ10378);江西省自然科学基金资助项目(20132BAB204019)

摘　　要：运用RT-PCR和RACE-PCR技术克隆了翘嘴鳜（Siniperca chuatsi）铜锌超氧化物歧化酶（命名为ScCu/Zn-SOD）基因,该基因的cDNA序列全长为804bp,开放阅读框为465bp,编码154个氨基酸。氨基酸序列中含2个Cu/Zn-SOD标签序列、7个铜锌金属结合位点、2个半胱氨酸位点和1个N-糖基化位点;其与胞内Cu/Zn-SOD（icCu/Zn-SOD）和胞外Cu/Zn-SOD（ecCu/Zn-SOD）氨基酸序列的同源性分别为60.13%~92.21%和33.77%~42.21%。在系统进化树中,ScCu/Zn-SOD与其它物种的icCu/Zn-SOD聚成一支。ScCu/Zn-SOD的蛋白晶体结构主要由2个α螺旋和8个β折叠组成,呈β折叠构象,通过结合1个Cu^2＋和1个Zn^2＋构成其酶活性中心。ScCu/Zn-SOD的mRNA在翘嘴鳜肌肉、鳃、肝脏和肾脏等组织中均有表达,且鳃中的相对表达量最高。将该基因编码蛋白序列与pET-30a表达载体重组,热激转入大肠杆菌BL21（DE3）感受态细胞中诱导表达,SDS-PAGE检测发现诱导后菌液的超声上清中有可溶性重组蛋白表达。The Copper/Zinc superoxide dismutase gene, designated as ScCu/Zn-SOD, was cloned from the mandarin fish Siniperca chuatsi by using the reverse transcription polymerase chain reaction and rapid am- plification of cDNA ends method. The full-length of cDNA gene consisted of 804 nucleotides,including an open-reading frame of 465 bp encoding a 154 amino acids protein. In the amino acid sequence of ScCu/Zn- SOD,two conservative signatures of Cu/Zn-SOD, seven active amino acid sites binding with Cu/Zn, two cysteine residues and one N-glycosylation sites were identified. Comparison ScCu/Zn-SOD with Cu/Zn- SOD from other species, ScCu/Zn-SOD showed high sequence identity （60.13 %- 92.21%） with icCu/Zn- SOD, but relatively low identity （33. 77%--42.21%） with the ecCu/Zn-SOD. In the Neighbor-joining phy- logentic tree of Cu/Zn-SOD amino acid sequences,ScCu/Zn-SOD got together ic Cu/Zn-SOD. The three di- mensional structure of ScCu/Zn-SOD protein mainly composed of two α-Helixs and eight 13-Sheets,and the enzyme activity center was constituted by binding one Cu^2＋ and one Zn^2＋. ScCu/Zn-SOD mRNA had widely expressed in all tissues, including muscle, gills, liver and kidney with the highest exnression level was ingills. Finally,the recombinant expression vector （ pET--30a＋ScCu/Zn-SOD ） was constructed,and had been induced after transformed into E. coli BL12 strains （DE3）. The result of protein gel electrophoresis （SDS-PAGE） showed that the soluble recombinant protein of ScCu/Zn-SOD was abundantly expressed in cell supernatant,which could be used for subsequent analysis of recombinant protein purification and anti- body production.

关 键 词：翘嘴鳜 超氧化物歧化酶 基因克隆 原核表达 

分 类 号：S944.1[农业科学—水产养殖] Q786[农业科学—水产科学]