摘要
Tth DNA聚合酶具有聚合酶与反转录酶两种功能,而且耐高温,但是国内并未实现高效生产.本研究以粗提的嗜热栖热菌Thermus thermophilus基因组为模板,采用PCR分段扩增和酶切连接的方法获得Tth DNA聚合酶基因全长序列;然后构建重组质粒pXT99b/Tth并转化表达菌E.coli DH5α,利用IPTG诱导Tth基因表达,并采用热处理、Ni2+柱纯化融合His-tag的Tth DNA聚合酶,最后利用PCR、RT-PCR鉴定其活性.结果显示,本研究获得Tth DNA聚合酶基因全长2 505bp,成功表达并获得高纯度高活性的重组Tth DNA聚合酶,1L发酵液所得粗酶液中,酶活性约有800 000U,酶的比活约为6 315U/mg.本研究最终实现了Tth DNA聚合酶的高效国产化并降低了实验成本.
Tth DNA polymerase is heat-resistant and has the two functions of polymerase and reverse transcriptase,but it has not been produced efficiently in China.In this study,the full-length sequence of Tth DNA polymerase gene was obtained from the genome of Thermus thermophilus by fractional amplification,enzyme digestion and ligation.Then it was recombined into pXT99 bexpression plasmid and transformed into E.coli DH5α,and expression of Tth DNA polymerase gene was induced by IPTG.Recombinant Tth DNA polymerase was purified by heat treatment and by Ni2+-resin affinity chromatograph,and its activity was identified by PCR and RT-PCR.The results show that the Tth full-length gene can be successfully obtained,and the recombinant Tth DNA polymerase is successfully expressed and purified.The total enzyme activity in 1 Lfermentation broth is about 800 000 U,and the specific enzyme activity is about 6 315 U/mg.In all,Tth DNA polymerase with high purity and high activity is efficiently localized and can reduce the cost of the experiment.
作者
李华
杨玲玲
杜红旗
LI Hua;YANG Lingling;DU Hongqi(College of Chemical Food,Zhengzhou Institute of Engineering Technology,Zhengzhou450044,China;Institute of Animal Husbandry and Veterinary Research,Henan Academy of Agricultural Sciences,Zhengzhou450008,China)
出处
《河南大学学报（自然科学版）》
CAS
2019年第3期311-317,共7页
Journal of Henan University:Natural Science
基金
河南省博士后科研项目启动经费资助(19030086)
