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

Generation of Selenium-containing Human Single-chain Fv Antibody with GPX Activity

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351U/μmol.

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（ll, 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-ll was purified by Ni^2＋ -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 Sees.