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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 sequence of fibrinolytic enzyme coding region of Eupolyphaga and to perform prokaryotic and eukaryotic expression. Methods: Primers were designed according to the published cDNA sequences of various animal plasmin genes. The coding region of Plasmodium vivax was cloned by RT-PCR and 3’RACE. The sequence was cloned into E. coli and Pichia pastoris expression. Results: Sequence analysis showed that the cloned plasminogen coding sequence was 672 bp in length, encoding a total of 224 amino acid residues. The initial amino acid sequence was IVGG, which was consistent with that of various animal plasmin. The cDNA sequence was expressed in Escherichia coli and Pichia pastoris, the former obtained expression of inactive protein, the latter obtained recombinant expression of fibrinolytic activity of the protein. Conclusion: The coding region of Plasmodium flexibensis was reported for the first time and was initially expressed, which lays the foundation for further study of its function.