【摘要】 根据乳酸乳球菌密码子的偏好性,在不改变编码蛋白的基础上,优化设计并合成枯草芽孢杆菌磷脂酰肌醇特异性磷脂酶C基因,将其与大肠埃希菌-乳酸乳球菌穿梭质粒p AMJ399连接,构建重组质粒p AMJ399-PIPLC,并电转化至乳酸乳球菌中进行诱导表达。SDS-PAGE分析显示:重组蛋白以可溶性蛋白的形式分泌于胞外,分子质量约35 ku,与预期蛋白大小一致。重组蛋白在PI-李斯特氏菌显色平板上显现明显的乳白色晕圈,证明重组蛋白具有酶活性,磷脂酰肌醇特异性磷脂酶C(PI-PLC)在重组乳酸乳球菌中成功获得表达。通过优化培养条件,以2%转接量,在含有1%红霉素抗性的GM17液体培养基中,于32℃静止培养24 h,测得培养基上清液中PI-PLC的浓度为1.092μg·m L-1。更多还原

【Abstract】 Based on the codon bias of Lactococcus lactis,phosphatidylinositol-specific phospholipase C gene from Bacillus cereus was optimized and then synthesized for protein expression. The cloned gene was inserted into Escherichia coli-Lactococcus lactis shuttle vector p AMJ399,and then transformed to L. lactis cells by electroporation to induce expression. The result of SDS-PAGE showed that the recombinant protein was secreted extracellular in the form of soluble proteins,and its molecular weight was about 35 ku,which was consistent with the expected protein size. Meanwhile,the recombinant protein showed significant enzyme activity on PI-Listeria chromogenic plate. The results indicated that phosphatidylinositol-specific phospholipase C( PI-PLC) was successfully expressed in L. lactis. The growth condition was optimized as follows: 2% inoculation amount; GM17 medium with 1% erythromycin; 32 ℃.After 24 h static culture under above condition,it could produce 1. 092 μg·m L- 1PI-PLC in the supernatant of culture medium.更多还原