假蕈状芽孢杆菌34kDa纤溶酶成熟肽基因的原核表达、纯化及活性分析  

Expression and Purification of Bacillus. pseudomycoides 34 kDa Fibrinolytic Enzyme Mature Peptide Gene and Activity Analysis

作　　者：刘慧娟[1] 郭晓军[1] 郭妍妍[1] 朱宝成[1] 

机构地区：[1]河北农业大学生命科学学院,河北保定071001

出　　处：《华北农学报》2011年第2期143-146,共4页Acta Agriculturae Boreali-Sinica

基　　金：河北省教育厅资助项目(2009123)

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34 kDa纤溶酶全长基因DNA序列(GenBank FJ463037)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40 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34 kDa纤溶酶成熟肽基因活性表达的重组菌。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-28a-G/BL21 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 fibrinolytic activity.As the result,we successfully obtained the recombinant bacteria in which 34 kDa fibrinolytic enzyme mature peptide gene expression with activity.

关 键 词：假蕈状芽孢杆菌 纤溶酶 成熟肽 表达 纯化 

分 类 号：Q786[生物学—分子生物学]