摘要
为彻底去除Taq DNA聚合酶中的大肠杆菌宿主DNA残留,旨在开发一种高效的表达与纯化策略,以满足分子生物学研究和临床应用对高纯度Taq DNA聚合酶的需求。采用pET-15b质粒在大肠杆菌BL21(DE3)中高效表达Taq DNA聚合酶,通过IPTG诱导实现目标蛋白的高水平表达,并结合Ni-NTA亲和层析与DEAE阴离子交换层析的纯化策略,实现对表达产物的高效纯化。结果表明:优化后的流程成功获得纯度超过95%的Taq DNA聚合酶;PCR检测大肠杆菌16S rRNA基因结果显示,自制Taq DNA聚合酶中未检出宿主DNA,其残留量与市售Taq DNA聚合酶相当。与传统的热失活结合硫酸铵沉淀或单一Ni-NTA亲和层析方法相比,优化的纯化流程能够更彻底地去除大肠杆菌宿主DNA残留,为高纯度Taq DNA聚合酶的制备提供了一种高效、可行的新策略。
In order to completely remove the residual host DNA from Escherichia coli in Taq DNA polymerase,theaim of this study was to develop an efficient expression and purification strategy to meet the demand for high-purityTaq DNA polymerase in molecular biology research and clinical application.The pET-15b plasmid was used for thehigh-efficiency expression of Taq DNA polymerase in Escherichia coli BL21(DE3),with IPTG induction ensuringthe high-level expression of the target protein.Then,the purification strategy combining Ni-NTA affinitychromatography and DEAE anion exchange chromatography was employed,in order to achieve the highly efficientpurification of the expressed product.The results showed that the optimized process successfully obtained Taq DNApolymerase with a purity of more than 95%.The results of PCR detection of Escherichia coli 16S rRNA gene showedthat the host DNA was not detected in the self-made Taq DNA polymerase,and the residual amount was equivalent tothat of the commercially available Taq DNA polymerase.Compared to the traditional thermal inactivation combinedwith ammonium sulfate precipitation or single Ni-NTA affinity chromatography,the optimized purification processcould more thoroughly remove the residual DNA of Escherichia coli host,which provided an efficient and feasiblenew strategy for the preparation of high-purity Taq DNA polymerase.
作者
吕新
刘兰英
李玥仁
罗土炎
LYU Xin;LIU Lan-ying;LI Yue-ren;LUO Tu-yan(Institute of Quality Standards&Testing Technology for Agro-Products,Fujian Academy of Agricultural Sciences,Fuzhou,Fujian 350003,China;Fujian Key Laboratory of Agro-products Quality&Safety,Fuzhou,Fujian 350003,China)
出处
《福建农业科技》
2025年第5期1-6,共6页
Fujian Agricultural Science and Technology
基金
福建省科技计划省属公益类专项(2021R1022007)
福建省财政专项—福建省农业科学院科技创新团队建设项目(CXTD2021011-3)
福建省人民政府、中国农业科学院“5511”协同创新工程(XTCXGC2021020)。
