论文部分内容阅读
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。
The half-antigen GSH-S-DNP was synthesized by the reaction of 2,4-dinitrochlorobenzene (DNCB) with the substrate glutathione (GSH) sulfhydryl group. The protein glutathione This hapten is conjugated to bovine serum albumin (BSA) to make the whole antigen. An average of 33 haptens were linked per BSA molecule as measured by absorption spectroscopy. BALB / c mice were immunized with whole antigen and hybridoma cell lines 4A4, 6A6, 1C8 and 4G3 of monoclonal antibody against GSH were prepared by lymphocyte hybridoma technique. 4A4IgG antibody was precipitated from 4A4 culture supernatant by 50% (NH4) 2SO4 precipitation, DEAE-52 cellulose ion exchange column chromatography and gel filtration on BiogelP-200. The purity of 4A4 IgG was more than 90% by SDS-PAGE. The serine (Ser) in 4A4IgG variable region is activated by phenylmethyl sulfonyl fluoride (PMSF) and then treated with hydrogen selenide (H2Se), then Ser is converted to selenocysteine (SeCys) and 4A4 IgG is introduced Catalytic gene. The chemically-mutated 4A4 IgG has glutathione peroxidase (GPX) activity, which is 1100 times more potent than the best GPX mimic PZ51 in the world and 2.2 x 104 times more free SeCys, Enzyme activity on the order of magnitude. After preliminary application, the antibody enzyme can prevent free radical damage to myocardial mitochondria.