甜菜夜蛾泛素延伸蛋白基因cDNA的3′RACE-PCR扩增及其克隆和表达  

Amplication with 3′RACE-PCR, cloning and prokaryotic expression of ubiquitin extension protein cDNA of Spodoptera exigua (Lepidoptera: Noctuidae)

作　　者：牛国栋[1] 张海元[1] 张忠信[1] 

机构地区：[1]中国科学院武汉病毒研究所分子病毒学重点实验室,武汉430071

出　　处：《昆虫学报》2004年第2期166-170,共5页Acta Entomologica Sinica

基　　金：国家"8 63"项目 ( 2 0 0 1AA2 460 14 );中国科学院农办重大项目 (NK十五 B 0 7 0 4)

摘　　要：根据已知的草地夜蛾Spodopterafrugiperda的泛素延伸基因 5′端核苷酸序列设计引物 ,应用 3′RACE PCR技术 ,从甜菜夜蛾S .exigua脂肪体组织总RNA中反转录扩增泛素基因的cDNA片段。扩增得到的片段全长 5 13bp ,3′末端有 12 3bp的非翻译区 ,翻译区编码一个长为 12 9个氨基酸残基的蛋白质 ,预测分子量为 14 8kD。同源分析表明 ,此cDNA序列为ubiquitin 5 3aaextensionprotein(ubi 5 3)基因 ,在泛素蛋白后融合了一个核糖体L4 0蛋白 (ribosomalL4 0protein)。用MagAlign和Genedoc软件对cDNA编码的氨基酸序列进行了同源性分析 ,结果表明 :甜菜夜蛾的ubi 5 3基因与真核生物家蚕Bombyxmori、草地夜蛾、果蝇Drosophilamelanogaster和人Homosapienes泛素的同源性分别为 96 9%、98 5 %、95 3%和 93 0 % ,与甜菜夜蛾核型多角体病毒(SeNPV)泛素的同源性为 78 8% ,说明真核生物的泛素基因与核型多角体病毒的泛素基因可能存在不同的分子进化途径。将甜菜夜蛾的ubi 5 3基因克隆到原核表达载体pET 2 8a上 ,转化至BL2 1(DE3)中 ,用IPTG进行诱导表达 ,用异源泛素单克隆抗体进行Westernblot检测 。To identify the nucleotide sequence of ubiquitin cDNA in Spodoptera exigua, a 513 bp cDNA fragment encoding ubiquitin-53aa extension protein from S. exigua lipid was amplified with 3′RACE (rapid amplification of cDNA ends) PCR with primers designed on the N-terminal amino acid sequence of S. frugiperda ubiquitin extension gene, and the amplified fragment was cloned and sequenced. The ubiquitin-53aa extension gene (ubi-53) in S. exigua was 513 bp in length, including 123 bp of 3′ untranslated region and 390 bp of encoding region. The encoding region encoded a peptide of 129 amino acid residues, in which there was an ubiquitin fused with a ribosomal L40 protein. A comparison of the deduced amino acid sequence with those of Bombyx mori, S. frugiperda, Drosophila melanogaster, Homo sapienes, SeNPV and BmNPV showed that the amino acid homology rates were 96.9%,98.5%,95.3%,93.0%, 78.8 % and 77.2% respectively. This suggests ube genes in eukaryotes may have a different evolution way from its host viruses. The fragment containing ubi-53 gene was inserted into pET-28a expressive vector, and the expression was induced by IPTG in E. coli BL21(DE3). The fusion protein was identified by Western blot using a mouse monoclonal antibody against bovine ubiquitin.

关 键 词：甜菜夜蛾 3′RACE—PCR 泛素延伸基因 分子进化 原核表达 

分 类 号：Q965[生物学—昆虫学]