意大利蜜蜂丙糖磷酸异构酶基因的克隆及其原核表达与纯化

从意大利蜜蜂Apis mellifera ligustica的肌肉组织中提取总RNA,采用RT-PCR的方法克隆蜜蜂第16号染色体上的丙糖磷酸异构酶基因的cDNA序列,将测序结果(GenBank登录号EU76098)与推导的氨基酸序列分别与GenBank中的其他物种进行同源比对分析。结果表明,该基因全长744bp,为完整的阅读框,编码247个氨基酸,成熟蛋白的理论分子量为26.89kD。比对结果显示AmTPI与家蚕、德国小镰、黄粉虫、丽蝇蛹集金小蜂、水稻等物种的基因相似性达69%以上,蛋白相似性达59%以上。将目的基因克隆到pGEX-4T-2融合表达载体上,并在大肠杆菌中得到成功表达,4h的表达量为总蛋白的42.1%。为了进一步探讨产物的酶学特性,实验还对表达产物进行纯化与浓缩。实验还构建增强型荧光真核表达质粒,为进一步研究AmTPI在真核细胞中的表达情况奠定基础。

Cloning, expression and purification of a recombinant triosephosphate isomerase from Apis mellifera ligustica

Total RNA was isolated from the muscles of honeybee, and the cDNA sequence of triosephosphate isomerase gene was then cloned by RT-PCR and sequenced (GenBank accession No. EU760983) . The gene was 744 bp in length, encoding 247 amino acids. The calculated molecular weight of the mature TPI protein was about 26.89 kD. The similarity between the AmTPI nucleotide sequence and those of Bombyx mori, Blattella germanica , Tenebrio molitor, Nasonia vitripennis and Oryza saliva TPI genes was over 69 % , but the similarity between their amino acid sequences was over 59% . The target gene was cloned to the pGEX-4T-2 vector GST fusion expression system. The results indicated that the fusion protein was expressed successfully in the E . Coli BL21 (DE3) cells and the amount of the expressed protein accounted for 42.1 % of the total proteins after 4 h induction. The recombinant fusion TPI protein was purified and concentrated to test its enzymatic properties. The brighter fluorescence recombinant EGFP vector is constructed for further study of expression in mammalian cells.