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北京大学学报（医学版） ›› 2019, Vol. 51 ›› Issue (3): 487-492. doi: 10.19723/j.issn.1671-167X.2019.03.016

• 论著 • 上一篇    下一篇

荧光分析法测定人体血液样品中脱嘌呤/脱嘧啶核酸内切酶1(APE1)的活性

王嘉禹,赵美萍△()   

  1. 北京分子科学国家研究中心,生物有机与分子工程教育部重点实验室,北京大学化学与分子工程学院,北京 100871
  • 收稿日期:2019-03-13 出版日期:2019-05-22 发布日期:2019-06-26
  • 作者简介:赵美萍,北京大学化学与分子工程学院教授,博士生导师。1990年、1993年、2002年先后获北京大学理学学士、硕士和博士学位。1997—1998年,荷兰阿姆斯特丹大学环境与毒理化学系访问学者;1998年,荷兰能源研究机构环境分析部访问学者。研究方向为分子识别与生物化学分析,近期主要围绕以下三个方面开展研究:(1)基因变化的高灵敏液体活检,包括基因突变、基因损伤和外源基因整合位点等的超高灵敏快速分析方法的建立及其在临床检验和兴奋剂检测方面的应用;(2)多种核酸修复酶的荧光分析、细胞成像和活性调控方法研究及其在癌症诊断和治疗中的应用;(3)基于分子印迹方法的新型纳米亲和材料的制备和识别机理研究及在生物分离和分析中的应用。现任国际分子印迹协会(Society of Mole-cular Imprinting,SMI)理事会成员、Applied Spectroscopy编委会成员、《分析科学学报》编委、中国分析测试协会标记免疫分析专业委员会常务委员、中国仪器仪表学会分析仪器分会样品制备专业委员会委员。迄今共主持国家自然科学基金7项,教育部博士点基金1项,国家高技术研究发展计划(863计划)课题1项,北京市自然科学基金2项。以第一作者或通信作者在Advanced Materials、Journal of the American Chemical Society、Nucleic Acids Research、Chemical Science、Analytical Chemistry等国内外重要学术期刊上发表研究论文120余篇,获授权发明专利10项。主编教材1部,培养博士研究生20余名。2018年获宝钢教育基金优秀教师奖。
  • 基金资助:
    国家自然科学基金(81571130100)、北大医学交叉研究种子基金(BMU2017MC008)-中央高校基本科研业务费和北京大学分子工程苏南研究院创新基金(SIM2018002848)

Fluorescence assay for the detection of apurinic/apyrimidinic endonuclease 1 (APE1) activity in human blood samples

Jia-yu WANG,Mei-ping ZHAO△()   

  1. Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
  • Received:2019-03-13 Online:2019-05-22 Published:2019-06-26
  • Supported by:
    Supported by the National Natural Science Foundation of China (81571130100), the Fundamental Research Funds for the Central Universities: Peking University Medicine Seed Fund for Interdisciplinary Research (BMU2017MC008), Innovation Fund of Sunan Institute for Molecular Engineering, Peking University (SIM2018002848)

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摘要： 目的 开发一种可快速、灵敏地检测生物样品中脱嘌呤/脱嘧啶核酸内切酶1(apurinic/apyrimidinic endonuclease 1,APE1)含量的荧光分析方法。方法 根据APE1所具有的脱碱基核酸内切酶活性,合成了一种用荧光基团与猝灭基团标记的含脱碱基位点的DNA荧光探针。在合适的缓冲体系下,APE1将该DNA探针水解并释放出荧光基团,根据荧光信号上升速率实现对APE1活性的定量检测。在此基础上,本研究改进了检测APE1时溶液缓冲液的条件,使得该荧光探针对APE1的响应更为灵敏,并进一步通过密度梯度离心法从人全血样品提取了外周血单核细胞(peripheral blood mononuclear cells,PBMCs),用改进后的荧光探针法定量测定了其蛋白提取液中APE1的含量。最后,使用该荧光探针法测定了临床血液样本中APE1的含量。结果 本研究建立的方法对APE1的最低检测限和功能灵敏度均为0.005 U/mL(3 pg/mL),线性范围为6 pg/mL~1.2 ng/mL。利用该方法测定了8份人血液样品中PBMCs蛋白中APE1的含量,测得每微克PBMCs蛋白中APE1的含量分布为0.061~0.40 ng,平均含量为0.16 ng APE1,加标回收率为98%±5%(n=3)。用该方法对102份正常人(男51例、女51例,年龄59~75岁)血清样品中的APE1含量进行了检测,得到这些血清样品中APE1含量的分布范围为0.13~0.34 ng/mL,加标回收率为96%±15%(n=3)。结论 本研究发展的荧光分析法操作简便、灵敏度高、所需生物样品量小,能够快速、准确地测定血液等生物样本中的APE1含量,解决了原有方法检测低含量血清样品时误差较大的问题,有良好的临床检验应用前景。

关键词: 光谱法, 荧光, 脱嘌呤/脱嘧啶核酸内切酶1, DNA探针, 血清, 外周血单核细胞

Abstract: Objective: To develop a simple, sensitive and robust method for rapid detection of human apurinic/apyrimidinic endonuclease 1 (APE1) in various biological samples.Methods: An abasic site-containing DNA probe with a sequence of 5'-T*T*C*C*T*C*T(ROX)AGAGXCGTT(BHQ2)C*A*C*T*G*T*AGTTTATA*C*A*G*T* GAATCTCTCTAG*T*C*T-3'[“X” represents AP site; The phosphorothioated nucleotides (at 3’ side) are indicated with an asterisk after the nucleotides; ROX is 6-carboxy-X-rhodamine and BHQ2 is Black Hole quencher 2] was synthesized and used for the detection. In the presence of APE1, the DNA probe could be specifically hydrolyzed by the enzyme and release the fluorophore, resulting in strong fluorescence emission. The activity of APE1 was determined according to the rate of increase in fluorescence intensity. In this work, we modified the reaction buffer and significantly improved the performance of the method. Moreover, the method was further extended to measure the contents of APE1 in the protein extraction from peripheral blood mononuclear cells (PBMCs) extracted from human whole blood samples by density gradient centrifugation. The assay was also applied to measure the activity of APE1 in human serum samples. Results: With a new reaction buffer composed of 0.04% (V/V) Triton X-100, 50 mmol/L KAc, 20 mmol/L Tris-Ac, 10 mmol/L Mg(Ac)2 and 1 mmol/L dithiothreitol (DTT), the method achieved a detection limit of 0.005 U/mL (3 pg/mL) and a linear response ranging from 6 pg/mL to 1.2 ng/mL. The contents of APE1 in the protein extraction from PBMCs of eight blood samples were measured to be in the range from 0.061 to 0.40 ng/μg protein, with an average of 0.16 ng/μg protein. The recovery was 98%±5% (n=3). The levels of APE1 in the sera from 102 normal individuals (51 male and 51 female, age range: 59-75 years) were observed to be from 0.13 to 0.34 ng/mL, with a recovery of 96%±15% (n=3).Conclusion: The new fluorescence assay was simple, rapid and sensitive, providing a practical tool to measure the activity of APE1 in serum samples and cell extracts. It also holds great potential in measurement of APE1 in many other biological samples for clinical test and laboratory research.

Key words: Spectrometry, fluorescence, Apurinic/apyrimidinic endonuclease 1, DNA probes, Serum, Peripheral blood mononuclear cells

中图分类号: 

  • R446.1

图1

A,在不同反应缓冲液中APE1探针(200 nmol/L)对相同浓度APE1(2.0 U/mL)响应的荧光曲线,其中NEB缓冲液4+0.04% Triton X-100为本研究新开发的反应缓冲液;B,在新反应液中APE1探针(300 nmol/L)与0.01~2.0 U/mL 范围内不同浓度 APE1反应的荧光曲线;C,在新反应液中APE1探针(300 nmol/L)与0.005~0.1 U/mL范围内不同浓度APE1反应的荧光曲线; D,在新反应液中检测APE1的线性范围"

图2

A,密度梯度离心法分离人PBMCs;B,不同稀释倍数测得原始血清中APE1的含量对照"

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ISSN 1671-167X
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