具有谷胱甘肽过氧化物酶活性的含硒单链抗体酶制备  

Generation of a Selenium-containing Single Chain Abzyme with Glutathione Peroxidase Activity

作　　者：由德林[1] 高姝娟[1] 黄华梁[2] 冯书章[3] 牟颖[1] 苏丹[1] 罗元明[1] 刘子[3] 闫岗林[1] 罗贵民[1] 杨同书[1] 沈家骢[4] 

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]中国科学院遗传研究所,北京100101 [3]中国人民解放军军需大学,长春130062 [4]超分子结构与谱学教育部重点实验室,长春130023

出　　处：《中国生物化学与分子生物学报》2001年第4期496-500,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家 8 6 3高技术研究发展计划项目(No:10 3 13 0 1 0 5 );国家科技部项目 (No .G2 0 0 0 0 7810 2 );国家自然科学基金会项目 (No .

摘　　要：利用RT PCR从分泌有谷胱甘肽结合部位的单克隆抗体杂交瘤细胞株 2F3中 ,扩增出单抗重链可变区和轻链可变区基因 .经DNA测序后 ,用Linker(Gly4 Ser1) 3 构建成单链抗体 (scFv)表达载体pTMF scFv ,将重组质粒pTMF scFv转化到大肠杆菌BL2 1(DE3) ,实现了单链抗体的高效表达 .表达的单链抗体占菌体总蛋白 2 5%～ 30 % .该重组蛋白以包涵体形式存在 ,分子量为 30kD .经过金属螯合亲和层析纯化、复性和凝胶过滤纯化 ,得到电泳均一的单链抗体 .再经化学诱变 ,得到含硒单链抗体酶 ,其谷胱甘肽过氧化物酶活性为 330 0U μmol.cDNA of heavy chain variable region and light chain variable region of monoclonal antibody 2F3 were successfully amplified from the hybridoma cells with RT\|PCR method.The heavy chain variable region gene was connected to the light chain variable gene by a linker(G 4S 1) 3 to obtain the gene of single chain fragment (scFv).The expression vector pTMF\|scFv was constructed and the scFv fragment was expressed in E.coli BL21(DE3) as inclusion bodies,and the expression level of scFv was about 25%—30% of the total proteins.The molecular weight of the recombinant protein scFv is 30 kD.Selenium\|containing scFv was obtained by Co 2+ \|IMAC affinity chromatography,renaturation and chemical mutation.The glutathione peroxidase activity of the selenium\|containing scFv was 3 300 U/μmol.The association constant of scFv for GSH was determined using fluorescence titration.

关 键 词：单链抗体 化学诱变 抗体酶 硒 谷胱甘肽过氧化物酶 

分 类 号：Q554[生物学—生物化学]