杨氏柠檬酸杆菌磷脂酶A1基因在E.coli中的表达

Molecular Clone and Expression of Phospholipase A1 Gene from Citrobacter youngae in Escherichia coli

DOI：10.3969/j.issn.1673-1689.2015.11.009

中文关键词: 杨氏柠檬酸杆菌 磷脂酶A1 表达 优化 大肠杆菌

英文关键词: Citrobacter youngae,phospholipase A1,expression,optimization,E.coli

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中文摘要:

为实现磷脂酶A1（PLA1）的异源表达，将杨氏柠檬酸杆菌（CICC No.21596）PLA1基因插入载体pET28a（+）中，构建重组表达质粒pET28a（+）-pla1，并将重组质粒转入宿主菌E.coli BL21（DE3）中，获得重组菌pET28a（+）-pla1/DE3。在IPTG诱导作用下经SDS-PAGE检测，发现在重组菌发酵破碎上清液中存在33 000大小的蛋白质，与预期蛋白质大小相符。在硼砂卵黄平板上对重组菌PLA1活性进行检测，结果显示重组菌具有明显的PLA1活性，表明PLA1基因在大肠杆菌中得到了表达。经发酵初步优化，获得摇瓶发酵的最佳诱导表达条件为：转接体积分数4%、诱导时机2 h、IPTG终浓度为0.4 mmol/L、37 ℃诱导培养8 h。经酸碱滴定法测得最高酶活为（5.6±0.2） U/mL。

英文摘要:

For the heterologous expression of phospholipase A1，the gene of phospholipase A1 from Citrobacter youngae（CICC No.21596） was cloned into the expression vector pET28a（+） to construct the recombinant plasmid pET28a（+）-pla1.Then the recombinant plasmid was transformed into E.coli BL21（DE3） to achieve the recombinant strain pET28a（+）-pla1/DE3. After subsequent induction by isopropyl β-D-1-thiogalactopyranoside （IPTG），an approximately 33 000 protein was detected in the cell lysate supernatant by SDS-PAGE. The PLA1 activity was detected in an egg yolk agar plate，indicating that the PLA1 gene was expressed in E.coli. The best induction conditions for PLA1 expression were achieved by the optimization of fermentation condition and it was showed as follows，4% of inoculum amount，2 h of initial culture，8 h of induced culture in the presence of 0.4 mmol/L of IPTG，and growth at 37 ℃. Under the optimized conditions，the maximum PLA1 activity in the supernatant was （5.6±0.2） U/mL.

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