化学突变具有底物结合部位的单克隆抗体制备含硒抗体酶

开发了一种制备抗体酶的新方法。用二硝基氯苯（ＤＮＣＢ）专一地与谷胱甘肽（ＧＳＨ）的巯基反应，合成出半抗原ＧＳＨ－Ｓ－ＤＮＰ。用戊二醛将半抗原偶联到牛血清白蛋白（ＢＳＡ）上，制成全抗原。再用标准的单抗制备法获得具有ＧＳＨ结合部位的单抗（４Ａ４ＩｇＧ）。用苯甲基磺酞氟（ＰＭＳＦ）和Ｈ２Ｓｅ相继处理该单抗，则将单拉结合部位上的丝氨酸（Ｓｅｒ）突变成硒代半胱氨酸（ＳｅＣｙｓ，因而在单抗结合部位上引入了谷胱甘肽过氧化物酶（ＧＰＸ）的催化基团。突变后的单抗具有ＧＰＸ活性，其活力已达到天然ＧＰＸ的数量级水平。动力学行为也与天然ＧＰＸ类似。这种新的含硒抗体酶有优于ＧＰＸ的一些特点。

Preparation of a Selenium-containing Abzyme by Chemical Mutation of the Monoclonal Antibody with Substrate Binding Sites

A new strategy for generating abzyme was developed.Thiol group of reduced glntathione(GSH) was specifieally reacted with 2, 4-dinitroclilorobenzene(DNCB)to give s-substituted dinitrophenyl glutathione as happen (GSH-S-DNP).The hapten was cross-linked to bovine serum albumin(BSA) by glutaraldehyde obtaining antigen.Then,the monoelonal antibody with GSH binding site(4A4IgG) was obtained by using monoelonal antibody preparation technique.The 4A4IgG was anccessively treated by phenylm ethylsulfonyl fluoride(PMSF) and hydrogen selenide(H2 Se) resulting in the conversion of serine residuesin the binding Bite of 4A4 IgG into selenocysteines,wineh are the catalgho groups of GPX.Thus,the mutated 4A4 IgG displayed GPX activity, winch approached the magnitude level of the native enzyme.The study on kinetic properties showed that both the abzyme and GPX exhibited similar kin6tie behavior.The abzyme has advantages over GPX in some respects.