Pfu DNA聚合酶的表达条件优化及纯化研究  

The Expression of Pfu DNA Polymerase Conditions Optimization and Purification Research

作　　者：欧阳乐军[1] 李莉梅[1] 布梁灏 刘雅丽 柳镜炬 黄嘉玲 蔡治全 赵俊仁[1] 倪燕妹 黄明超 Ouyang Lejun;Li Limei;Bu Lianghao;Liu Yali;Liu Jinju;Huang Jialing;Cai Zhiquan;Zhao Junren;Ni Yanmei;Huang Mingchao(Guangdong University of Petrochemical Technology,Maoming,525000;Agricultural Reclamation Tropical Crop Scientific Research Institute of Guangdong,Maoming,525000)

机构地区：[1]广东石油化工学院,茂名525000 [2]广东农垦热带作物科学研究所,茂名525000

出　　处：《基因组学与应用生物学》2019年第12期5458-5464,共7页Genomics and Applied Biology

基　　金：广东省大学生攀登计划项目(pdjhb0338; pdjhb0343)及重点项目(pdjh2019b0323);国家级大学生创新创业训练计划项目及校级培育项目;国家自然科学基金(31470677);广东省自然科学基金(2017A030307017);广东科技计划项目(201-7A030303087)共同资助

摘　　要：Pfu DNA聚合酶是分子生物学研究中最常用的高保真DNA聚合酶之一。本研究对重组菌株的Pfu酶基因表达条件进行优化,以提高Pfu DNA聚合酶的表达效率。通过在菌液深度OD600=0.87时加入1.5 mmol/L的IPTG进行诱导培养,诱导培养时间为11.03 h,用酶溶法对重组菌株进行破胞提取粗酶液,经热变性法与盐析沉淀法对杂酶进行纯化,最后经过透析后得到纯酶。采用考马斯亮蓝G-250法对酶进行含量测定及SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测纯度,最后使用PCR反应检测提纯后的Pfu酶的活性。结果表明优化处理后制备的Pfu DNA聚合酶,其纯度、酶活性和酶特异性均达到市售的Pfu DNA聚合酶水平。本研究为Pfu DNA聚合酶的开发和利用奠定了基础。Pfu DNA polymerase is one of the most commonly used high fidelity DNA polymerases in molecular biological research.In this study,the culture conditions of recombination strain were optimized to improve expression efficiency of Pfu DNA polymerase.The optimized culture conditions were as follows,1.5 mmol/L IPTG was added to the culture medium to induce culture when the OD600 of the bacterial solution is 0.87 and the culture time was 11.03 hours.The crude enzyme was extracted by enzymatic dissolution of the recombination strain.The enzyme was purified by thermal denaturation and salting-out.The purified enzyme was obtained by dialysis.Moreover,the content of the enzyme was determined by Coomassie brilliant blue G-250 method,examined purity by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),and the activity of purified Pfu DNA polymerase was detected by polymerase chain reaction(PCR).The results revealed that the Pfu DNA polymerase which was prepared and optimizing process by this method could reach commercial level of Pfu DNA polymerase in purity,enzyme activity and enzyme specificity.This study lays a foundation for the development and utilization of Pfu DNA polymerase.

关 键 词：高保真 DNA聚合酶 热变性 PCR反应 

分 类 号：Q55[生物学—生物化学]