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[摘要]
本文在大肠杆菌(E.coli BL21 DE3)中分别克隆表达了产气荚膜梭菌(Clostridium perfringens)和单核细胞增生性李斯特菌(Listeria monocytogenes)来源的磷脂酶C(PLC)基因,并系统研究了内源表达的重组PLC对大肠杆菌BL21(DE3)内外膜通透性的影响。结果表明内源表达的产气荚膜梭菌和单核细胞增生性李斯特菌重组PLC对宿主菌内膜通透性影响较大,但对宿主菌外膜通透性的影响相对较弱。此外,本文对两种不同来源重组表达的磷脂酶C蛋白进行了分离纯化,分析了外源添加重组磷脂酶C对大肠杆菌 BL21(DE3)细胞膜通透性的影响。结果表明外源PLC对大肠杆菌外膜通透性影响较大,而对内膜通透性影响相对较小。综上所述,产气荚膜梭菌和单核细胞增生性李斯特菌来源的磷脂酶C可以改善大肠杆菌细胞膜的通透性,为进一步提高大肠杆菌工程菌株蛋白异源表达提供新的思路。
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[Abstract]
The phospholipase C proteins from Clostridium perfringens(Cp-PLC)and Listeria monocytogenes (lm-PLC) were cloned and expressed in E. coli BL21 (DE3), and the effect of PLC on the permeability of E. coli BL21 membranes was investigated systematically. The recombinant PLCs expressed in C. perfringens and L. monocytogenes clearly affected the permeability of the inner membrane In contrast, the permeability of the outer bacterial membrane increased slightly. In addition, the effect of PLC enzymes incorporation on the permeabilization of E. coli BL21 membranes was tested, indicating that the addition of PLC enzymes markedly increased the permeabilization of the outer membrane. However, the permeabilization of bacterial inner membrane was not affected to the same extent. More importantly, coexpression of Cp-PLC and lm-PLC in E. coli BL21 to increase permeabilization of the host cell membrane opens a promising strategy for enhancing recombinant protein expression.
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