具有谷胱甘肽过氧化物酶（GPX）活力的抗体酶研究──单克隆抗体的制备及化学诱变

Study on Abzyme with Activity of Glutathione Peroxidase(GPX) Preparation and Chemical Mutation of Monoclonal Antibodies

将２，４－二硝基氯苯（ＤＮＣＢ）专一地与底物谷胱甘肽（ＧＳＨ）巯基反应，合成了半抗原ＧＳＨ－Ｓ－ＤＮＰ；通过蛋白质连接技术（戊二醛法）将此半抗原偶联到牛血清白蛋白（ＢＳＡ）上，制备出全抗原。用吸收光谱测得每个ＢＳＡ分子上平均连接３３个半抗原。用全抗原免疫ＢＡＬＢ／ｃ小鼠，通过淋巴细胞杂交瘤技术，制备出抗ＧＳＨ的单克隆抗体杂交瘤细胞系４Ａ４、６Ａ６、１Ｃ８和４Ｇ３等。将其中的４Ａ４培养上清经５０％（ＮＨ４）２ＳＯ４沉淀、ＤＥＡＥ－５２纤维素离子交换柱层析和ＢｉｏｇｅｌＰ－２００凝胶过滤分离得到４Ａ４ＩｇＧ抗体，经ＳＤＳ－ＰＡＧＥ检验纯度大于９０％。４Ａ４ＩｇＧ可变区中的丝氨酸（Ｓｅｒ）经苯甲基磺酰氟（ＰＭＳＦ）活化，再用硒化氢（Ｈ２Ｓｅ）处理，则Ｓｅｒ转变成硒代半胱氨酸（ＳｅＣｙｓ），使４Ａ４ＩｇＧ引入该催化基因。化学诱变后的４Ａ４ＩｇＧ具有谷胱甘肽过氧化物酶（ＧＰＸ）活性，其活力是世界上最好的ＧＰＸ模拟物ＰＺ５１的１１００倍，是游离ＳｅＣｙｓ的２．２×１０４倍，已达到天然酶活力的数量级水平。经初步应用，该抗体酶可防止自由基对心肌线粒体的损伤。

Thiol group of reduced glutathione (GSH) concentratedly reacted with 2,4-dinitrochlorobezene (DNCB) to give S-substituted dinitrophenyl glutathione as hapten (GSH-SDNP). The hapten was cross-linked to amino groups on the surface of bovine serum albumin (BSA) by glutaraldehyde to obtain whole antigen. By using absorption spectrum method,it was detected that about 33 of the GSH-S-DNP haptens were linked to every BSA molecule.The BALB/c mice were immunized with the whole antigen GSH-S-DNP-BSA,and hybridoma cell lines 4A4, 6A6, 1C8 and 4G3 which secreted monoclonal antibodies specific to GSH were established by lymphocyte hybridoma technique. The supernatant of 4A4 was precipitated by 50%saturated (NH4)2 SO4,further pruifed by DEAE-52 cellulose ion-exchange and Biogel P-200 gel chromatography. with SDS-PAGE, it was detected that the purity of 4A4 IgG antibody was above 90%.The serine residues in V-domains of 4A4 IgG antibody were activated by phenylmethylsulfonyl fluoride (PMSF), and treated by hydrogen selenide (H2Se), resulting in conversion of serine into selenocysteine(SeCys), leading catalytic group into 4A4 IgG antibody. The mutated 4A4 IgG4 displayed GPX catalytic activity, which was 1100 times more than that of PZ51, the best GPX mimic known in the world, and 2.2×104 times more than free SeCys, approaching the magnitude level of native enzyme activity.Used preliminarily,the 4A4 lgG Abzyme could prevent free radicals doing harm to myocardial mitochondria.These results indicated that the combination of cell engineering and technology of biochemistry was a handy and effective way to prepare catalytic antibodies-Abzymes.