高等学校化学学报

高等学校化学学报

• 研究论文 • 上一篇    下一篇

量子点与人源抗谷胱甘肽单链抗体的连接与表征

徐俊杰1, 王诗雯1, 赵虹4, 陈桂秋5, 霍锐1, 田莉1, 段玉晶1, 李敏杰3, 杨柏3, 魏景艳1,2
  

    1. 吉林大学药学院,
    2. 生命科学学院, 分子酶学工程教育部重点实验室, 长春 130021;
    3. 吉林大学化学学院, 超分子结构与材料国家重点实验室, 长春 130012;
    4. 吉林大学中日联谊医院中心实验室,
    5. 病理科, 长春 130031
  • 收稿日期:2008-06-20 修回日期:1900-01-01 出版日期:2009-03-10 发布日期:2009-03-10
  • 通讯作者: 魏景艳

Preparation and Characterization of Quantum Dots-anti-GSH Human scFvs Bioconjugations

XU Jun-Jie1, WANG Shi-Wen1, ZHAO Hong4, CHEN Gui-Qiu5, HUO Rui1, TIAN Li1, DUAN Yu-Jing1, LI Min-Jie3, YANG Bai3, WEI Jing-Yan1,2*
  

    1. College of Pharmaceutical Science,
    2. Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, College of Life Science, Jilin University, Changchun 130021, China;
    3. State Key Lab for Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, China;
    4. Central Laboratory,
    5. Pathology Department, China-Japan Union Hospital, Jilin University, Changchun 130031, China
  • Received:2008-06-20 Revised:1900-01-01 Online:2009-03-10 Published:2009-03-10
  • Contact: WEI Jing-Yan

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被引次数

9

摘要： 用已构建的表达载体pPELB-B3, 在大肠杆菌Rosetta中可溶性表达人源抗谷胱甘肽(GSH)单链抗体B3(scFv-B3), 经Ni2+螯合亲和层析纯化后, 用点印迹法验证了其与GSH结合的特异性. 将水相合成的半导体纳米粒子(半导体量子点, QDs)在N-羟基琥珀酰亚胺(NHS)和1-乙基-3-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)的作用下, 与scFvs连接. 光谱分析和膜印迹结果表明, scFvs成功地共价连接到QDs表面, 所得的QD-scFvs复合物能够较好地识别GSH. 荧光显微镜观察QD-scFvs与人乳腺癌细胞MCF-7的作用结果, 初步判断QD-scFvs能够跨膜进入细胞.

关键词: 量子点, 人源单链抗体, 谷胱甘肽过氧化物酶(GPX), 共价结合, 印迹

Abstract: In order to generate catalytic antibodies with glutathione peroxidase(GPX) activity, the clone B3 that bound specifically to glutathione(GSH) was selected from the phage display antibody library[human synthetic VH+VL single-chain Fv fragment(scFv) library] and the expression vector of scFv-B3 was constructed in previous study. The expression vector pPELB-B3 constructed was transformed into the Escherichia coli Rosetta to express the human anti-GSH single chain fragments variable(scFvs) antibodies. The scFv-B3 was purified by Ni2+-immobilized metal affinity chromatography(IMAC). 3-Mercaptopropyl acid-stabilized CdTe quantum dots synthesized in aqueous solution were used to conjugate with scFvs. Spectra analysis show that the fluorescent of QD-scFvs undergo a blue-shift in the emission peak, which suggest that the scFvs have been effectively bound to the scFvs, via covalent conjugation. It is also suggested by the result of the simple new method of dot blot. The results of membrane dot indicate that the QD-scFvs can specifically recognize the GSH. The status of the QD-scFvs acting on the MCF-7 cells was observed under fluorescence microscope. The results show that QD-scFvs can enter the cytoplasm across the membrane.

Key words: Quantum dot, Human single chain fragments variable(scFvs), Glutathione peroxidase(GPX), Covalent conjugation, Dot blot

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