应用蛋白质工程法制备含硒单链抗体酶  

Preparation of A Selenium-containing Single-chain Abzyme with Glutathione Peroxidase Activity by Protein Engineering

作　　者：牟颖[1] 高姝娟[1] 张岩[1] 苏丹[1] 苏大明[1] 任晓君[1] 由德林[1] 罗元明[1] 闫岗林[1] 罗贵民[1] 黄华梁[2] 刘子[3] 

机构地区：[1]吉林大学分子酶学工程教育部重点实验室,长春130023 [2]中国科学院遗传学研究所,北京100101 [3]中国人民解放军军需大学,长春130062

出　　处：《中国生物制品学杂志》2001年第2期87-90,共4页Chinese Journal of Biologicals

基　　金：863高技术(103-13-01-05);国家自然科学资助(20072010)。

摘　　要：目的 制备一种具有谷胱甘肽过氧化物酬（ＧＰＸ）活性的含硒单链抗体酶。方法 利用ＲＴ－ＰＣＲ方 法从杂交瘤细胞株２Ｆ３中扩增出单克隆抗体重链可变区和轻链可变区基因。经ＤＮＡ序列测定后，构建成表达载体 ｐＴＭＦ－ｓｃＦｖ，经过金属螯合层析纯化、复性和化学诱变，得到含硒单链抗体酶。结果 将重组质粒ｐＴＭＦ－ｓｃＦｖ分别转 化入大肠杆菌ＪＭ１０９（ＤＥ３）、ＢＬ２１（ＤＥ３）和 ＢＬ２１（ｃｏｄｅｎ　ｐｌｕｓ），表达目的蛋白分别占菌体总蛋白的５％－１０％、１５％－ ２０％和 ２５％－３０％。该重组蛋白以包涵体形式存在，相对分子质量为３００００。其 ＧＰＸ活性为３４００Ｕ／μｍｏｌ，接近天 然酌水平。结论 为工业化制备含硒单链抗体酶奠定基础。Objective To prepare a selenium-containing single-chain abzyme with glutathione peroxidase(GPX)activity.Methods The genes were amplified from the heavy(VH)and light(VL)chain variable regions of hybridoma cell strain 2F3 by RT-PCR and identified by DNA sequencing. The recombinant plasmid pTMF-scFv was constructed with the genes and transformed to E. coli ,and expressed product was purified by Co2+ -IMAC affinity chromatography. A selenium-containing single-chain abzyme with glutathione peroxidase activity was obtained by the renaturation and chemical mutagenesis of the purified expressed product. Results SDS-PAGE showed that the expressed scFv existed in the form of inclusion body with a molecular weight of 30000.The scFv expressed in E. coli .1M109(DE3) ,B121(DE3)and BL21(coden plus)contained about 5% - 10% ,15% - 20% and 25% - 30% of total protein respectively.The GPX activity of the selenium-containing single-chain abzyme( 3400U/μmol) was close to that of natural GPX enzyme. Conclusion The study laid a foundation of industrial production of selenium-containing single-chain abzyme with glutathione peroxidase activity.

关 键 词：单链抗体酶 包涵体 谷胱甘肽过氧化物酶 硒 化学突变 蛋白质工程法 

分 类 号：Q814[生物学—生物工程] Q554.6