摘要
目的:在大肠杆菌中高效表达重组Pfu酶。方法:将pfu基因构建于载体pET28a上,重组质粒pET28a-pfu转化DH5α,获得了含pET28a-pfu重组表达菌株。在重组菌株OD600为0.2时,经终浓度为1 mmol/L的IPTG诱导表达10～12 h后,菌体超声波破壁后,80℃条件下热变性30 min去除部分杂蛋白;粗酶液再经Ni离子亲和层析进一步纯化。结果:获得了高纯度的重组Pfu酶,利用该酶成功扩增出目的基因片段。结论:纯化的Pfu酶具有较高的活性,比活性为62 500 U/mg,该研究表达的重组酶完全可以替代商用Pfu酶。
Objective ：To highly expression of Pfu in E. coli. Method：pET28a- pfu was successfully constructed by inserting pfu gene into pET28a,the recombinant DI-ISct was obtained by transforming pET28a - pfu into E. coli DHSct. When OD600 of recombinant DH5a was 0. 2, pfu was inducibly expressed by using IPTG at the final concentration of 1 mmol/L 10 - 12 h later, recombinant DH5 a pellet was heat- ed at 80℃ for 30 rain after wall - broken by ultrasonic, The extraction was furified by metal - affinity chromatography on Ni2 ＋ - Sepharosecolumns. Result：High -quality -purified Pfu was obtained ,The pfu DNA polymerase activity was confirmed by successf Lly amplification of a target DNA through PCR. Conclusion：It was confirmed that the Pfu enzyme was highly expressed and characterized by high DNA pol- ymerase activity.
出处
《生物技术》
CAS
CSCD
北大核心
2012年第5期29-31,共3页
Biotechnology
