纳豆激酶基因在大肠杆菌中的表达及活性分析

目的:构建纳豆激酶基因的表达载体,鉴定其在大肠杆菌中的表达及表达产物的生物活性鉴定.方法:以纳豆芽胞杆菌基因组为模板,PCR技术克隆出纳豆激酶基因的成熟肽序列,分别克隆进具有信号肽的pMAL-p2x及无信号肽pMAL-c2x质粒中,经酶切和测序鉴定其正确性,分别将重组质粒转化至大肠杆菌中表达.结果:成功构建的两组重组质粒在IPTG诱导下,均能分别在37℃及16℃条件下表达出可溶性的融合蛋白,SDS-PAGE胶检测证实重组质粒在大肠杆菌中可表达出相对分子量约76kDa的纳豆激酶蛋白.纤维蛋白平板实验证明两种融合蛋白均有活性,且有信号肽的融合蛋白的酶活较无信号肽的融合蛋白高.结果:成功构建了两组重组纳豆激酶基因的表达质粒,且该两组重组基因在大肠杆菌中可溶性表达并具有生物活性,因此为下一步研究表达产物纳豆激酶的功能、应用和生产奠定了基础.

Secretory Expression of Nattokinase from Bacillus natto in Escherichia coli

Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. A novel strategy to express a nattokinase from Bacillus natto in a live delivery vehicle E. coli BL21(DE3) was developed. The mature peptide-coding fragment of NK gene was amplified from B. natto total DNA by polymerase chain reaction (PCR). The sequence analysis indicated that the fragment of NK gene consisted of 825 bp, which encoded 275 amino acid residues. The sequence of NK gene has 99% homology with those of the reported ones. The expression plasmids NK-pep-pMAL-p2X and NK-pep-pMAL-c2X are constructed by inserting NK gene into vector pMAL-p2X and pMAL-c2X, and then fusionally expressed with maltose-binding protein (MBP) in Escherichia coli BL21(DE3). SDS-PAGE analysis revealed that the NK protein were secretion expressed after induction with IPTG for 4 hours under 16℃ or 37℃.The characteristic of this recombinant nattokinase is comparable to the native nattokinase. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.