天冬酰胺酶胞外表达的初步研究

目的]为降低L-天冬酰胺酶Ⅱ的生产成本提供依据。方法]通过PCR反应扩增大肠杆菌JM109成熟区的L-天冬酰胺酶II基因(ansB),将扩增片段与pMD18-T载体连接,构建重组质粒pMD18-T-asp。对pMD18-T-asp与质粒pET-22b进行双酶切后,将它们连接起来,转化感受态细胞,筛选重组表达载体pET-22b-asp。利用重组表达载体pET-22b-asp转化BL21(DE3),并对其发酵条件以及分离、纯化工艺进行了初步研究。结果]成功克隆长度为960 bp的L-天冬酰胺酶II基因。重组表达载体pET-22b-asp也构建成功,并在BL21中得到表达。在重组菌接入TB培养基后2 h加入IPTG、培养基的初始pH值为7.2、装液量为12%,L-天冬酰胺酶Ⅱ酶活最高。重组天冬酰胺酶的纯化收率为82%,比活力达196 U/mg。结论]重组天冬酰胺酶的胞外表达大大简化了其分离步骤。

Preliminary Study on the Extracellular Expression of Asparaginase

Objective] The aim of the research was to provide the basis for reducing the production cost of L asparaginase Ⅱ.Method] L-asparaginase II gene(ansB) in JM109 maturation zone of Escherichia coli was amplified through PCR reaction.The amplified fragment was connected with pMD18-T vector to construct the recombinant plasmid pMD18-T-asp.After double-enzyme digestion of pMD18-T-asp and plasmid pET-22b,they were connected to transform the competent cells and screen out the recombinant expression vector pET-22b-asp which was used to transform BL21(DE3).And its fermentation conditions,the separation and purification process were preliminarily studied.Result] L-asparaginase II gene with the length of 960 bp was successfully cloned.The recombinant expression vector pET-22b-asp was also successfully constructed and it was expressed in BL21.When IPTG was added after inoculating the recombinant bacteria on TB medium for 2 h with the initial medium pH value of 7.2 and liquid volume in flask of 12%,the enzyme activity of L-asparaginase II was highest..The purification yield of the recombinant asparaginase was 82% and the specific activity reached 196 U/mg.Conclusion] The extracellular expression of the recombinant asparaginase simplified its separation steps greatly.