谭拥军,吴莎莎,谭桂湘,向勤.Bst DNA聚合酶的纯化及等温扩增检测体系的构建[J].湖南大学学报:自然科学版,2019,(12):98~106
Bst DNA聚合酶的纯化及等温扩增检测体系的构建
Purification of Bst DNA Polymerase and Construction of Isothermal Amplification Detection System
  
DOI:
中文关键词:  Bst DNA 聚合酶  重组蛋白  扩增  核酸检测
英文关键词:Bst DNA polymerase  recombinant proteins  amplification  nucleic acid detection
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作者单位
谭拥军,吴莎莎,谭桂湘,向勤 (湖南大学 生物学院湖南 长沙 410082) 
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中文摘要:
      Bst DNA 聚合酶是等温扩增反应中的一个关键酶,但由于专利限制,目前国内实验室所使用的Bst DNA 聚合酶大多需要从国外进口,价格昂贵且长途运输影响酶活性,因此亟需自主生产出Bst DNA 聚合酶应用于等温扩增检测技术. 通过构建Bst Fragement的原核表达质粒,采用原核蛋白表达系统和His-Tag亲和纯化手段,制备成本低,活性高,特异性强的Bst DNA 聚合酶. 同时,针对特定的模板,对自制Bst DNA聚合酶的扩增反应进行了进一步优化,用不同的产物鉴定方法对扩增效果进行检测,并最终筛选出该酶最优的扩增反应条件为60 mM K+、30 mM (NH4)2SO4、pH为 9.0. 最后,验证了Bst DNA聚合酶能用于快速准确检测肺炎支原体、肺炎衣原体,降低了相关病原体核酸检测的成本,为之后核酸检测在基层医疗的更广泛应用提供了条件.
英文摘要:
      Bst DNA polymerase is a key enzyme in isothermal amplification reaction. However, due to the patent restrictions, most of the Bst DNA polymerase used in domestic laboratories need to be imported abroad, which is expensive and needs long distance transportation. Therefore, it is urgent to produce Bst DNA polymerase for isothermal amplification detection technology independently. From constructing a plasmid containing Bst Fragment, Bst DNA polymerase was prepared on a large scale by prokaryotic expression system followed by His-tag affinity purification method. The purified Bst DNA polymerase possessed the advantages such as low cost, high activity, and strong specificity in the isothermal amplification reaction. The activity of Bst DNA polymerase was optimized for specific DNA templates by detecting the products of amplification. At the same time, the optimal amplification reaction conditions of the enzyme were finally selected,which were 60 mM K+,30 mM(NH4)2SO4,and pH 9.0. With reference to the relevant patents, the obtained Bst DNA polymerase was able to detect mycoplasma pneumonia and Chlamydia pneumonia quickly and accurately through isothermal amplification reactions. In conclusion, a low cost enzyme was provided for isothermal amplification detection of related pathogen DNA, which can be potentially used in the primary medical care.
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