野桑蚕铜锌超氧化物歧化酶基因的克隆分析与原核表达  

Cloning,Analysis and Expression of Copper/Zinc Superoxide Dismutase From Wild Silkworm,Bombyx mandarina

作　　者：丛海峰[1] 徐世清[1] 司马杨虎[1] 张升祥[2] 王更先[1] 董航 刘莹[1] 柳学广[1] 

机构地区：[1]苏州大学生命科学学院,苏州215123 [2]山东农业大学林学院,泰安271018 [3]溧阳市蚕桑技术指导站,常州213300

出　　处：《蚕业科学》2007年第2期234-240,共7页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(编号30371086);国家重点基础研究发展计划"973"项目(编号2005CB121005)

摘　　要：超氧化物歧化酶是野桑蚕保护酶体系的重要酶类。利用RT-PCR方法克隆出野桑蚕铜锌超氧化物歧化酶(Cu/Zn-SOD)cDNA(EMB I登陆号:AM410997),其开放阅读框ORF长465 bp,编码154个氨基酸。同源性及系统进化分析表明,推导出的氨基酸序列与3种果蝇的同源性平均为69.3%,与线虫为57%,各物种中与Cu/Zn结合的残基高度保守。利用ExPASy的ScanProsite以及PSort和TMpred对此编码的蛋白质结构和功能域分析,预测出其具有2段Cu/Zn-SOD特异序列,且该蛋白不存在信号肽以及跨膜区。将Cu/Zn-SOD cDNA克隆到pET-28 a(+)表达载体,测序鉴定后以IPTG诱导表达,SDS-PAGE电泳鉴定其表达的融合蛋白质分子量为19.4 kD。Superoxide dismutase （SOD） is an important antioxygenic enzyme in Bombyx mandarina. This study focused on Copper/Zinc superoxide dismutase （Cu/Zn-SOD） from Bombyx mandarina. cDNA encoding Cu/Zn-SOD was firstly amplified by RT-PCR （ EMBL accession number. AM410997）. The sequence of cDNA indicated that the open reading frame comprises 465 base pairs encoding 154 amino acid residues. The deduced amino acid sequence of Cu/Zn-SOD showed 69.3% identity with those of three species of Drosophila on average, 57% identity with that of Caenorhabditis elegans, respectively. The structure and functional domain of Cu/Zn-SOD was analyzed according to the software of ScanProsite, PSort and TMpred on line. The results showed that the protein contains two conservative motifs of Cu/Zn-SOD, and has no signal peptide or transmembrane region. The gene was cloned into expressing vector pET-28a（＋） and expressed. SDS-PAGE analysis showed that the target protein was expressed at a high level and the putative molecular weight of recombinant protein was 19.4 kD.

关 键 词：野桑蚕 铜锌超氧化物歧化酶 序列分析 原核表达 

分 类 号：S885.9[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]