Pfu-sso7d DNA聚合酶的制备及反应缓冲液的研究  

Preparation of Pfu-sso7d DNA polymerase and study of reaction buffer

作　　者：刘俊伶 陶倩倩 李琳钰 姚新欣 张鑫 LIU Jun-ling;TAO Qian-qian;LI Lin-yun;YAO Xin-xin;ZHANG Xin(Gannan Innovation and Translational Medicine Research Institute,Gannan Medical University,Ganzhou,Jiangxi 341000)

机构地区：[1]赣南医科大学赣南创新与转化医学研究院,江西赣州341000

出　　处：《赣南医学院学报》2024年第4期374-382,共9页JOURNAL OF GANNAN MEDICAL UNIVERSITY

摘　　要：目的:制备有高效扩增长片段以及复杂片段能力的Pfu DNA聚合酶。方法:运用基因工程学方法构建重组质粒pET-28a-Pfu-sso7d,采用优化后的诱导条件对pET-28a-Pfu-sso7d实行诱导表达,将菌液超声破碎并提取上清液,应用镍亲和层析洗脱纯化取得较纯Pfu-sso7d DNA聚合酶。采用qPCR方法对酶进行酶活力单位定量、确定反应缓冲液组分最佳浓度及检测自制Pfu-sso7d DNA聚合酶酶活性。结果:在1 mmol·L^(-1) IPTG、16℃条件下诱导Pfu-sso7d DNA聚合酶菌液16 h,Pfu-sso7d DNA聚合酶高效表达;用20~100 mmol·L^(-1)咪唑可将蛋白洗脱。Pfu-sso7d DNA聚合酶最终活力单位定量为1 U·μL^(-1)。PCR最佳反应缓冲液为pH9.0、20 mmol·L^(-1) Tris-HCl、0.6 mmol·L^(-1) MgSO_(4)、50 mmol·L^(-1) KCl、5 mmol·L^(-1)(NH_(4))_(2)SO_(4)、0.1%Triton X-100,添加剂组分为3%DMSO、1 mol·L^(-1)甜菜碱。Pfu-sso7d DNA聚合酶能高效扩增3000 bp的目的条带,活性达到商用高保真DNA聚合酶水平。结论:基于基因重组制备的Pfu-sso7d DNA聚合酶可进行高效的PCR反应,节省了实验室PCR成本,为进一步提高Pfu DNA聚合酶活性提供一定技术参考。Objective:To prepare Pfu DNA polymerase with high efficiency amplification fragment and complex fragment ability.Methods:Firstly,the recombinant plasmid pET-28a-Pfu-sso7d was constructed by genetic engineering,and the optimized induction conditions were used to induce the expression of pET-28a-Pfu-sso7d.After that,the cells were broken by ultrasonic smashing method and the crude enzyme solution was extracted.Then,the Ni affinity chromatography was used to purify the purified Pfu-sso7d DNA polymerase,and the enzyme activity unit was quantified by qPCR.Secondly,the optimal concentration of reaction buffer was determined by PCR reaction.Finally,the activity of the self-made Pfu-sso7d DNA polymerase was detected by PCR reaction.Results:After induction with 1 mmol·L^(-1) IPTG for 16 h at 16℃,Pfu-sso7d DNA polymerase was expressed efficiently;when the concentration of imidazole was 20-100 mmol·L^(-1),the protein could be eluted.The final activity unit of Pfu-sso7d DNA polymerase was quantified as 1U·μL^(-1).When the PCR reaction system was pH 9.0,20 mmol·L^(-1) Tris-HCl,0.6 mmol·L^(-1) MgSO_(4),50 mmol·L^(-1) KCl,5 mmol·L^(-1)(NH_(4))_(2)SO_(4),0.1%Triton X-100,and the additive composition was 3%DMSO,1 mol·L^(-1) betaine,the target band of 3000 bp could be amplified efficiently,and the complex fragment could be amplified efficiently,and the activity could reach the level of commercial high-fidelity DNA polymerase.Conclusion:Pfu-sso7d DNA polymerase prepared based on genetic recombination can be used for efficient PCR reactions.The preparation saves the laboratory PCR cost and provides a theoretical reference for the further development and utilization of Pfu DNA polymerase.

关 键 词：聚合酶链式反应 Pfu DNA聚合酶 Sso7d 反应缓冲液 

分 类 号：R3[医药卫生—基础医学]