人pcsk9基因原核表达载体的构建及诱导表达
发布时间：2018-09-14 13:16
【摘要】：目的构建人前蛋白转化酶枯草溶菌素9(pcsk9)的原核表达载体,优化pcsk9诱导表达条件。方法聚合酶链式反应扩增人工合成的pcsk9 cDNA序列。扩增产物经双酶切、连接,构建pET-28b-pcsk9表达载体;将重组质粒转化入大肠杆菌表达菌BL21(DE3)的感受态细胞中,分别在不同条件下对其进行诱导,获得pET-28b-pcsk9的最佳诱导表达条件。结果成功构建了pET-28b-pcsk9重组表达载体,其最佳表达条件为菌液光密度值取0.6,诱导温度取30℃,诱导剂终浓度取1.0 mmol/L,诱导时间取8 h。结论该重组表达载体构建成功,在大肠杆菌表达菌BL21(DE3)中可有效地表达,对诱导所用的温度和时间相对比较敏感。
[Abstract]:Objective to construct the prokaryotic expression vector of human proprotein converting enzyme lysogenin 9 (pcsk9) and to optimize the pcsk9 induced expression conditions. Methods the synthetic pcsk9 cDNA sequence was amplified by polymerase chain reaction. The amplified product was digested by double enzyme and ligated to construct the pET-28b-pcsk9 expression vector. The recombinant plasmid was transformed into the competent cells of Escherichia coli expression strain BL21 (DE3) and induced under different conditions to obtain the best induced expression conditions of pET-28b-pcsk9. Results the recombinant expression vector of pET-28b-pcsk9 was successfully constructed. The optimal expression conditions were as follows: the optical density of the liquid was 0.6, the induction temperature was 30 鈩，
本文编号：2242817
[Abstract]:Objective to construct the prokaryotic expression vector of human proprotein converting enzyme lysogenin 9 (pcsk9) and to optimize the pcsk9 induced expression conditions. Methods the synthetic pcsk9 cDNA sequence was amplified by polymerase chain reaction. The amplified product was digested by double enzyme and ligated to construct the pET-28b-pcsk9 expression vector. The recombinant plasmid was transformed into the competent cells of Escherichia coli expression strain BL21 (DE3) and induced under different conditions to obtain the best induced expression conditions of pET-28b-pcsk9. Results the recombinant expression vector of pET-28b-pcsk9 was successfully constructed. The optimal expression conditions were as follows: the optical density of the liquid was 0.6, the induction temperature was 30 鈩，
本文编号：2242817
本文链接：https://www.wllwen.com/yixuelunwen/jichuyixue/2242817.html
