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.
出处
《蚕业科学》
CAS
CSCD
北大核心
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)
关键词
野桑蚕
铜锌超氧化物歧化酶
序列分析
原核表达
Bombyx mandarina
Copper/Zinc superoxide dismutase
Sequence Analysis
Prokaryotic expression
