高等学校化学学报 ›› 2015, Vol. 36 ›› Issue (3): 511-515.doi: 10.7503/cjcu20140803

• 有机化学 • 上一篇    下一篇

水相合成荧光纳米晶与碱性蛋白酶的偶联及纯化

祝金明2, 孙卓1, 付瑶1, 郭轶1, 徐力1()   

  1. 1. 吉林大学生命科学学院分子酶学工程教育部重点实验室, 长春 130012
    2. 吉林大学中日联谊医院, 长春 130033
  • 收稿日期:2014-09-04 出版日期:2015-03-10 发布日期:2015-02-04
  • 作者简介:联系人简介: 徐 力, 女, 博士, 教授, 博士生导师, 主要从事酶与纳米生物技术研究. E-mail: xuli@jlu.edu.cn
  • 基金资助:
    国家自然科学基金(批准号: 81271697)、 高等学校博士学科点专项科研基金(批准号: 20120061110021)和吉林省科技发展计划项目(批准号: 20106031, 20120967, YYZX201264, 20130206069GX)资助

Labeling and Purification of Alcalase and Fluorescent Nanocrystals Synthesized in Water Phase

ZHU Jinming2, SUN Zhuo1, FU Yao1, GUO Yi1, XU Li1,*()   

  1. 1. Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education,College of Life Science, Jilin University, Changchun 130012, China
    2. China-Japan Union Hospital of Jilin University, Changchun 130033, China
  • Received:2014-09-04 Online:2015-03-10 Published:2015-02-04
  • Contact: XU Li E-mail:xuli@jlu.edu.cn
  • Supported by:
    † Supported by the National Natural Science Foundation of China(No.81271697), the Specialized Research Fund for the Doctoral Program of Higher Education of China(No.20120061110021) and the Social Development Project of Science and Technology Department of Jilin Province of China(Nos.20106031, 20120967, YYZX201264, 20130206069GX)

摘要:

利用水相中合成的巯基丙酸包覆的CdTe荧光纳米晶与碱性蛋白酶(Alcalase)进行偶联标记. 荧光光谱分析表明, CdTe纳米晶体具有较窄的粒度分布和较高的光稳定性, 室温下30 d时荧光强度降低8.36%. 透射电子显微镜结果显示, CdTe纳米晶的直径约为3 nm; X射线衍射结果表明, CeTe纳米晶具有闪锌矿结构. 利用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺磺酸钠盐(NHSS)偶联方法将CdTe纳米晶与Alcalase进行偶联, 通过超滤离心、 凝胶柱层析及凝胶电泳技术对偶联后的CdTe-Alcalase进行分离和纯化. 荧光光谱显示偶联产物具有很好的光稳定性和较高的酶活力(61.06 U/μg). 在荧光显微镜下可观察到标记蛋白的荧光, 为研究酶反应的示踪及酶的构效关系等提供了实验基础.

关键词: 荧光纳米晶, 碱性蛋白酶, 偶联标记, 纯化

Abstract:

CdTe nanocrystals were synthesized in aqueous solution and capped with amercaptopropionic acid, which were used as a kind of fluorescent probe to label Alcalase. The fluorescence spectra showed that CdTe nanocrystals were accompanied by a narrow size distribution and high stability. The fluorescence intensity decreased 8.36% after 30 d at room temperature. Transmission electron microscopy(TEM) imaging revealed that CdTe nanocrystal’s diameter was about 3 nm, and X-ray diffraction(XRD) pattern showed it with zinc blende structure. After being conjugated with Alcalase using 1-ethyl-3-(3-dimethyl aminopropye) carbodii-mide hydroclloride(EDC) and N-hydroxysulfosucinimide sodium salt(NHSS) methods, the CdTe-Alcalase conjugates were separated and purified by ultra-filter-centrifugation, a Sephadex G-100 column and sodium dedecye sulfate(SDS)-polyacrylamide gel electrophoresis. Fluorescent spectral analysis showed that CdTe-Alcalase conjugates also presented good optical stability compared with CdTe nanocrystals. The enzyme activity was also relatively high at 61.06 U/μg. The fluorescent emission of the CdTe-Alcalase conjugates was visualized with a fluorescence microscope, which would be suitable for tracing the enzyme reaction in the future.

Key words: Fluorescent nanocrystal, Alcalase, Labeling, Purification

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