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| 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 | |
| 引用本文： | 霍锐,石毅,魏景艳,徐俊杰,吕绍武,闫飞,苏家明,段玉晶,王诗雯,丛登立,李唯,闫岗林,罗贵民.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[J].高等学校化学学报,2008,29(7):1379-1383. |
| 作者姓名： | 霍锐 石毅 魏景艳 徐俊杰 吕绍武 闫飞 苏家明 段玉晶 王诗雯 丛登立 李唯 闫岗林 罗贵民 |
| 作者单位： | 1. 吉林大学药学院,长春,130021;吉林大学分子酶学工程教育部重点实验室,长春,130021 2. 吉林大学药学院,长春,130021 3. 吉林大学分子酶学工程教育部重点实验室,长春,130021 4. 吉林大学生命科学学院,长春,130021 |
| 基金项目： | 吉林省科学技术厅科研基金 , 长春市科学技术局科研基金 |
| 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 |
| 收稿时间： | 2008-03-11 |
Generation of Selenium-containing Human Single-chain Fv Antibody with GPX Activity |
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| HUO Rui,SHI Yi,WEI Jing-Yan,XU Jun-Jie,Lu Shao-Wu,YAN Fei,SU Jia-Ming,DUAN Yu-Jing,WANG Shi-Wen,CONG Deng-Li,LI Wei,YAN Gang-Lin,LUO Gui-Min.Generation of Selenium-containing Human Single-chain Fv Antibody with GPX Activity[J].Chemical Research In Chinese Universities,2008,29(7):1379-1383. | |
| Authors: | HUO Rui SHI Yi WEI Jing-Yan XU Jun-Jie Lu Shao-Wu YAN Fei SU Jia-Ming DUAN Yu-Jing WANG Shi-Wen CONG Deng-Li LI Wei YAN Gang-Lin LUO Gui-Min |
| Institution: | College of Pharmaceutical Science, ;Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, ;College of Life Science, Jilin University, Changchun 130021, China |
| 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. |
| Keywords: | Human scFv Phage display antibody library GPX Selenium Chemical mutation |
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