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

具有谷胱甘肽过氧化物酶活性的含硒人源单链抗体的制备

霍锐1,2, 石毅1, 魏景艳1,2, 徐俊杰1, 吕绍武2, 闫飞2, 苏家明3, 段玉晶1, 王诗雯1, 丛登立1, 李唯3, 闫岗林2, 罗贵民2   

    1. 吉林大学药学院,
    2. 分子酶学工程教育部重点实验室,
    3. 生命科学学院, 长春 130021
  • 收稿日期:2008-03-11 修回日期:1900-01-01 出版日期:2008-07-10 发布日期:2008-07-10
  • 通讯作者: 魏景艳

Generation of Selenium-containing Human Single-chain Fv Antibody with GPX Activity

HUO Rui1,2, SHI Yi1, WEI Jing-Yan1,2*, XU Jun-Jie1, L

    1. College of Pharmaceutical Science,
    2. Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education,
    3. College of Life Science, Jilin University, Changchun 130021, China
  • Received:2008-03-11 Revised:1900-01-01 Online:2008-07-10 Published:2008-07-10
  • Contact: WEI Jing-Yan

PDF (PC)

被引次数

8

摘要： 以谷胱甘肽(GSH)为靶抗原, 从噬菌体展示人源单链抗体库中筛选人源单链抗体(scFv). 经3轮筛选后, 用ELISA方法检测出5个(2, 11, 16, 24, 32 )可以和GSH结合的克隆. PCR产物的电泳和测序结果表明, 只有3个克隆(11, 16, 24)具有完整的scFv编码基因. 选取和GSH结合力高的克隆11的scFv 编码基因组装到表达载体pPELB上, 在大肠杆菌Rosetta中进行可溶性表达, 用Ni2+螯合亲和层析纯化scFv-11, 免疫点印迹结果证实该抗体能与GSH特异结合. 通过化学突变将scFv-11的丝氨酸转变成硒代半胱氨酸(Sec)后, 获得了具有谷胱甘肽过氧化物酶(GPX)活力的含硒(Se)人源单链抗体(Se-scFv-11), 其活力为351 U/μmol.

关键词: 人源单链抗体, 噬菌体展示抗体库, 谷胱甘肽过氧化物酶, 硒, 化学突变

Abstract: Human single chain fragments variable(scFvs) against GSH were screened from a phage display human scFv antibody library. After three rounds of panning, five clones(2, 11, 16, 24, 32 ) binding to GSH were selected by ELISA. Analysis of PCR products using gel electrophoresis and sequencing showed that three clones(11, 16, 24) contained intact scFv-encoding gene. The scFv-encoding gene from clone 11 was subcloned into the expression vector pPELB and expressed as soluble form(scFv-11) in Escherichia coli Rosetta. The scFv-11 was purified by Ni2+-immobilized metal affinity chromatography. The specificity to GSH of scFv-11 was verified by immuno-dot blot. Selenium-containing human scFv(Se-scFv-11) with GPX activity of 351 U/mol was obtained by the chemical modification of the reactive serine residues into Secs.

Key words: Human scFv, Phage display antibody library, GPX, Selenium, Chemical mutation

中图分类号: 

TrendMD: