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 MgSO4、50 mmol·L-1 KCl、5 mmol·L-1(NH4)2SO4、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3 000 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.
Abstract
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 mmo·l L-1 Tris-HCl,0.6 mmo·l L-1 MgSO4,50 mmo·l L-1 KCl,5 mmo·l L-1(NH4)2SO4,0.1%Triton X-100,and the additive composition was 3%DMSO,1 mol·L-1 betaine,the target band of 3 000 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.
维普
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