摘要： 为进一步探讨全长基因和成熟肽基因的关系,根据已发表的假蕈状芽孢杆菌3 kDa纤溶酶全长基因DNA序列(GenBank FJ63037)和纤溶酶活性位点的位置,设计合成一对引物,扩增得到纤溶酶成熟肽基因G(951 bp编码317个氨基酸),连接pET-28a构建pET-28a-G载体,热激转化大肠杆菌BL21,成功获得pET-28a-G⁄BL21工程菌.终浓度为1 mmol⁄L的IPTG诱导表达,经SDS-PAGE检测表达在胞内的融合蛋白,融合蛋白表观分子量约为0 kDa,符合所预测的结果.诱导菌体超声破壁后用纤维蛋白平板法检测表达产物具有纤溶酶活性,镍柱亲和层析纯化后的目的产物仍保留纤溶活性.本研究成功得到了3 kDa纤溶酶成熟肽基因活性表达的重组菌.

关键词: 假蕈状芽孢杆菌, 纤溶酶, 成熟肽, 表达, 纯化

Abstract: To further explore the relationship of gene between the full peptide and the mature peptide,we designed and synthesized a pair of primers according to DNA sequence(GenBank FJ463037)of B.pseudomycoides 34 kDa fibrinolytic enzyme published and fibrinolytic enzyme active site.We proformed PCR from the recombinant plasmid pGEX-BpFE and obtained fibrinolytic enzyme mature peptide gene-G(951 bp encoding,317 amino acid)to construct pET-28a-G vector.The recombinant plasmid of pET-28a-G was transformed into host bacterium of E.coli BL21 to construct pET-28 a-G/BL2 1 engineering strains successfully.SDS-PAGE analysis was performed after pET-28a-G/ BL21 being induced by IPTG(1 mmol/L)to detect intracellular expression of the fusion protein.Fusion protein had an apparent molecular weight of about 40 kDa,which was consistent with the predicted results.Fibrin-Plate method showed that the intracellular protein had activity,and end products after nickel affinity chromatography purified retained fibrinolie activity.As the result,we successfully obtained the recombinant bacteria in which 34 kDa fibrinolytic enzyme mature peptide gene expression with activity.

Key words: B．pseudomycoides, Fibrinolytic enzyme, Mature peptide, Expression, Purification

中图分类号: 

• Q786