具有谷胱甘肽过氧化物酶活性的含硒单链抗体酶制备

 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.采用荧光滴定法测定了单链抗体对谷胱甘肽的结合常数

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

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.