重组flap核酸内切酶1的表达及活性测定方法的建立

盛楠1 马寅姣1 王建平1 邹秉杰2 周国华1,3,4

(1.中国药科大学生命科学与技术学院, 江苏南京 210009)
(2.南京军区南京总医院药理科, 江苏南京 210002)
(3.南京军区南京总医院药理科, 江苏南京 210009)
(4.Department of Pharmacology,Nanjing General Hospital, Nanjing ,Jiangsu,China 210002)
【知识点链接】古细菌; 重组蛋白; 层析; 探针

【摘要】Flap核酸内切酶1(Flap endonuclease 1,FEN1)是一种能催化核酸侵入反应的核酸内切酶,可应用于信号放大检测方法,但该酶详细的表达纯化工艺尚无报道,并且活性难以准确测定,限制了其应用。通过合成嗜热古球菌Archaeoglobus fulgidus来源的FEN1基因序列,构建了p ET24a(+)-FEN1-His重组质粒,并通过优化表达条件,得到了FEN1最优表达条件为:37℃、200 r/min振荡培养8 h后,加入诱导剂IPTG至终浓度为0.05 mmol/L,再于37℃、200 r/min诱导表达11 h,最终经镍亲和层析成功纯化得到了分子量约为38 k Da的重组FEN1。同时建立了基于荧光标记探针的FEN1活性测定方法,准确测定了重组FEN1的活性,为建立基于该酶的核酸检测方法提供了可靠的酶活力依据。最终将重组FEN1用于实时荧光PCR偶联高特异核酸侵入信号扩增法检测了乙醛脱氢酶2基因(aldh2)的基因型,得到了准确的分型结果,表明重组FEN1能用于基因多态性的分型检测中,为发展基于核酸侵入反应的核酸检测方法提供了可靠的工具酶。

【关键词】 flap核酸内切酶1; 信号放大; 活性测定;

【DOI】

【基金资助】 国家科技重大专项(No.2013ZX10004103) National Key Science&Technology Special Project(No.2013ZX10004103) 江苏省临床科技专项(No.BL2012061) Jiangsu Province’s Clinical Science&Technology Special Project(No.BL2012061) 国家自然科学基金(Nos.31300704,21475151,21405176,21275161) National Natural Science Foundation of China(Nos.31300704,21475151,21405176,21275161) 中国博士后基金特别资助项目基金(Nos.2013T60938,2014T71011) China Postdoctoral Science Special Foundation(Nos.2013T60938,2014T71011) 江苏省六大人才高峰项目(No.2015-WSN-085)资助 Six Talent Peaks Project in Jiangsu Province(No.2015-WSN-085)

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This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 10, Pages 1433-1442

October 2016

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摘要

  • 1 材料与方法
  • 2 结果与分析
  • 3 结论
  • 参考文献