摘要
为了建立一套基于大肠杆菌表达系统的嵌合式高保真DNA聚合酶的重组表达与纯化的工艺,首先将含有目的基因的质粒转化至大肠杆菌BL21中,通过高压破碎仪进行细胞破碎,再用离子交换层析方法获得嵌合型高保真聚合酶。优化反应条件显示在28℃自动诱导下表达的嵌合DNA聚合酶蛋白产量较高。利用高压破碎仪将细胞破碎获得可溶蛋白,然后利用硫酸铵沉淀将蛋白粗纯,并采用离子交换层析柱纯化,最终获得较纯的DNA聚合酶。通过对获得的嵌合DNA聚合酶进行活性分析,证实该聚合酶具有高效的活性,为以后大规模生产高保真DNA聚合酶奠定基础。
The purpose of this experiment is to establish a process for recombinant expression and purification of a chimeric high-fidelity DNA polymerase using the E.coli expression system.Following the established protocol for producing high-fidelity polymerase in E.coli,the plasmid containing the target gene was initially transformed into E.coli BL21 strain.The target polymerase protein was then expressed using an incubator.Subsequently,the cells were crushed using a high-pressure homogenizer,and the target protein was obtained through ion exchange chromatography.The results demonstrated that the chimeric DNA polymerase protein exhibited a higher yield when expressed at 28C under auto-induction condition.The Soluble protein was acquired using a high-pressure homogenizer,and subsequent ammonium sulfate precipitation enabled the primary purification of target protein.Additionally,an ion exchange chromatography column was utilized to obtain the pure DNA polymerase.Through the activity analysis of the obtained chimeric DNA polymerase,it was confirmed that the polymerase exhibited high activity.This preparation serves as a foundation for future large-scale production of high-fidelity DNA polymerase.
作者
王尧
王玉玲
张闻慧
吴银荣
赵梦然
罗忠宜
林森
WANG Yao;WANG Yuling;ZHANG Wenhui;WU Yinrong;ZHAO Mengran;LUO Zhongyi;LIN Sen(College of Biology and Food Engineering,Anyang Institute of Technology,Anyang 455000,China;Surgical Diagnostics Pty Ltd,Roseville,Sydney 2069,Australia;Anyang Kindstar Global Medical Laboratory Ltd,Anyang 455000,China)
出处
《安阳工学院学报》
2023年第4期120-124,共5页
Journal of Anyang Institute of Technology
基金
河南省科技计划(222102310210)
安阳市科技发展计划(2021A01SF002)
安阳工学院博士后启动基金(BSJ2020017,BSJ2021030)。
