重组大肠杆菌表达铜绿假单胞菌溶血性磷脂酶C

Expression of hemolytic phospholipase C from Pseudomonas aeruginosa in Escherichia coli

摘要:

摘要:【目的】构建产溶血性磷脂酶C(Hemolytic Phospholipase C，PLCH)的重组大肠杆菌(Escherich coli)菌株，并初步优化其发酵条件。【方法】首先利用卵黄硼砂平板分离法筛选到一株产磷脂酶C(Phospholipase C，PLC)活性较高的菌株，命名为铜绿假单胞菌(Pseudomonas aeruginosa)41;进一步以P.aeruginosa 41基因组DNA为模板设计引物，PCR扩增获得溶血性磷脂酶C(PLCH)基因，构建重组大肠杆菌表达质粒并转化大肠杆菌E.coli BL21(DE3);筛选转化子并检测PLC活性和溶血能力，并初步优化其发酵条件。【结果】成功构建了重组大肠杆菌E.coli BL21(DE3)/pET28a-plcH;在硼砂卵黄平板上对重组菌进行PLC活性测定，显示重组菌有明显的磷脂酶C活性;在哥伦比亚血琼脂平板上对重组菌进行溶血性试验，表明PLCH具有较强的溶血活性;初步优化摇瓶发酵条件为:5%转接量，37℃、200 r/min下培养4 h添加IPTG至终浓度为0.9 mmol/L，转为25℃、150r /min诱导培养14 h;优化后重组菌的酶活可达到722.89±0.47 U/mL。【结论】本文成功构建了一株产溶血性磷脂酶C活性较高的重组大肠杆菌菌株，并通过优化发酵条件使其酶活达到了722.89±0.47 U/mL，本实验在国内首次实现了铜绿假单胞菌来源的溶血性磷脂酶C基因在大肠杆菌的胞内表达，该研究为研究磷脂酶C产业化奠定了一定的基础。

Abstract:

Abstract:［Objective］In this study，we constructed a recombinant Escherichia coli (E.coli) strain to produce hemolytic phospholipase C and optimized the fermentation conditions．［Methods］ We screened a high phospholipase C activity strain，Pseudomonas aeruginosa (P.aeruginosa) 41，through yolk borax plate method，and cloned the hemolytic phospholipase C gene (plcH) from it．The plcH was inserted into pET-28a (+) and then obtained the recombinant expression plasmid (pET28a-plcH)．We selected the correct recombinant plasmid and transformed it into E.coli BL21 (DE3)．Furthermore，we determined the PLC activity and hemolytic activity in positive transformants on yolk borax plate and columbia blood agar plate．Finally，we optimized the fermentation conditions. [Results］We successfully constructed a recombinant E．coli strain (E．coli BL21 (DE3)/pET28a-plcH) that showed significant phospholipase C activity．Moreover，hemolytic phospholipase C of the recombinant strain showed strong hemolytic activity． The enzyme activity of phospholipase C was 722.9±0.47 U/mL with 5% of inoculation amount，200 r/min for 4 hours at temperature of 37，induced by 0.9 mmol/L IPTG for 14 hours．［Conclusion］ We constructed a recombinant E．coli strain with high hemolytic phospholipase C activity under optimized fermentation conditions． It is the first time in domestic to successfully clone and express phospholipase C gene from P．aeruginosa in E．coli．These research results are helpful to advance the industrialization and application of phospholipase C．