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

根据已知的草地夜蛾Spodoptera frugiperda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513 bp，3'末端有123 bp的非翻译区，翻译区编码一个长为129个氨基酸残基的蛋白质，预测分子量为14.8 kD。同源分析表明，此cDNA序列为ubiquitin-53aa extension protein(ubi-53) 基因，在泛素蛋白后融合了一个核糖体L40蛋白（ribosomal L40 protein）。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-53基因与真核生物家蚕Bombyx mori、草地夜蛾、果蝇Drosophila melanogaster和人Homo sapienes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-53基因克隆到原核表达载体pET-28a上，转化至BL21（DE3）中，用IPTG进行诱导表达，用异源泛素单克隆抗体进行Western blot检测，证明原核表达蛋白是目的蛋白。

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

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 biquitin 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, includeing 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.