DNA结合蛋白DbpA影响DNA聚合酶性能的研究  

Research of Impact of DNA Binding Protein DbpA on the Performance of Pfu DNA Polymerase

作　　者：曹淑中 裘丽珍[1] 

机构地区：[1]复旦大学生命科学学院,上海200438

出　　处：《复旦学报（自然科学版）》2015年第4期469-477,F0003,共10页Journal of Fudan University：Natural Science

摘　　要：DNA聚合酶是催化DNA合成的酶,在体内和体外都具有重要的作用.在体内DNA聚合酶参与DNA的复制、损伤修复等,在体外DNA聚合酶是现代分子生物学重要的工具酶.Pfu DNA聚合酶因具有高保真性而被广泛使用,但延伸速度慢,提高其延伸速度将使其应用更加广泛.DbpA是古生菌Sulfolobus solfataricus细胞内含量丰富的非特异性双链DNA结合蛋白,分子质量8ku.本研究通过引物重叠延伸的方法得到密码子优化的DbpA基因,并构建得到Pfu-DbpA基因.结果表明:融合有DbpA蛋白的Pfu DNA聚合酶(Pfu-DbpA DNA聚合酶)自身的热稳定性不受影响,而延伸速度得到了提高;同时具有更高的耐盐性,说明具有更高的持续合成能力,并且能够适应更广泛的缓冲液体系.应用Pfu-DbpA DNA聚合酶扩增λDNA不同长度的片段,结果显示其比Pfu DNA聚合酶具有更好的应用效果.DNA polymerase is the enzyme catalyze the synthesis of DNA, and plays important role both in vivo and in vitro. DNA polymerase involves in DNA replication and damage repair in vivo, and is an important tool enzyme used in modern molecular biology research. Pfu DNA polymerase has been widely used for its high fidelity, but its extension rate is low. It would allow extensive application with improved extension rate of Pfu DNA polemerase. DbpA is an 8 ku non-specific .double-stranded DNA binding protein abound in archaeabacteria Sulfolobus solfataricus. In this research, the＋eodon optimized DbpA gene has been constructed by oligo overlapping extension and fused to construct Pfu-DbpA gene. The results indicate that DbpA does not affect the thermo stability of Pfu DNA polymerase, but improve the extension rate and salt tolerance of Pfu DNA polymerase. Higher salt tolerance indicate higher processivity and would allow Pfu DNA polyrnerase function under more widely buffer systems. Pfu- DbpA DNA polymerase perform better than Pfu DNA polymerase in amplifying different lengths of λDNA.

关 键 词：PFU DNA聚合酶 DBPA 延伸速度 

分 类 号：Q816[生物学—生物工程]