摘要
[Objective] The paper was to improve the preparation efficacy of Taq DNA polymerase. [Method] Ni column was used to purify Taq DNA polymerase carrying with 6xHis tag,and recombined vector. Using the thermal-resistant characteristics of Taq DNA polymerase,the crude extract was treated at 75 ℃ for 1 h,and the activity of prepared enzyme solution was verified by PCR test. [Result] The recombinant pET-32A-Taq could highly express in BL21 （DE3） host bacteria and remove hybrid protein by thermal denaturation. The enzyme preparation with the activity further higher than purchased Taq DNA polymerase was obtained. [Conclusion] Taq DNA polymerase prepared by thermal purification method is simple with low cost,and can meet the needs of a large number of conventional PCR amplification.
[目的]提高Taq DNA聚合酶的制备效率。[方法]利用Ni柱亲和色谱纯化载有6xHis标记的Taq DNA聚合酶,并重组载体,利用Taq DNA聚合酶的耐热特性,对粗提液75℃处理1h,之后通过PCR试验验证制备酶液的活力。[结果]所获重组的pET-32A-Taq能够在BL21(DE3)宿主菌中高效表达并可通过热变性去除杂蛋白,获得了活力远高于购买的Taq DNA聚合酶的酶制剂。[结论]使用热纯化法制备的Taq DNA聚合酶工艺简单,成本较低,能满足常规大量PCR实验要求。
基金
Supported by National Natural Science Foundation of China (30872254)~~
