应用蛋白质工程法制备含硒单链抗体酶

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。

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

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.