土鳖虫纤溶酶编码区序列的克隆与表达

Cloning and expression of fibrinolytic enzyme cDNA sequence from Eupolyphaga sinensis

目的:获得土鳖虫纤溶酶编码区序列,并进行原核和真核表达。方法:根据已报道的多种动物纤溶酶基因cDNA序列设计引物,用RT-PCR和3′RACE法克隆得到土鳖虫纤溶酶编码区序列;将该序列克隆进入大肠杆菌和毕赤酵母进行表达。结果:序列分析表明,所克隆的纤溶酶编码区序列长672bp,共编码224个氨基酸残基,起始氨基酸序列为IVGG,与多种动物纤溶酶一致。将此cDNA序列在大肠杆菌和毕赤酵母中进行表达,前者获得没有活性的表达蛋白,后者获得具有纤溶活性的重组表达蛋白。结论:首次报道了土鳖虫纤溶酶编码区序列,并进行了初步表达,为进一步研究其功能奠定了基础。

Objective：To obtain the cDNA sequence encoding fibrinolytic enzyme from Eupolyphaga sinensis and express it in prokaryotic and eukaryotic expression system.Method：The primers were designed according to the cDNA of other animals＇ fibrinolytic enzyme.The cDNA sequence was cloned by RT-PCR and 3＇RACE.Result：Sequence analysis revealed that the length of the cDNA fragment was 672 bp and encoded a protein of 224 amino acid residues,the N end amino acid sequence residues was IVGG in accordance with other fibrinolytic enzyme.The cDNA sequence was expressed in E.coli,inactive protein was obtained.While expressed in Pichia pastoris,recombinant protein had fibrinolytic activity.Conclusion：The cDNA sequence of fibrinolytic enzyme from E.sinensis Walker was cloned and expressed for the first time and it proved a good basis for further functional study of the enzyme.