人源含硒单链抗体模拟谷胱甘肽过氧化物酶

发布时间：2018-10-29 08:58

【摘要】： 含硒谷胱甘肽过氧化物酶(GPX)是机体内最重要的抗氧化酶,已有许多GPX的人工模拟酶,但活力普遍不高。对已有的GPX模拟酶进行深入研究表明,产生底物结合位点是制备高活力GPX模拟酶的关键因素之一。单克隆抗体(McAb)制备技术是产生具有底物结合部位受体的有效手段。罗贵民小组在制备模拟GPX抗体酶的过程中,提出了疏水修饰理论并应用该理论制备了一系列具有较高GPX活力的单克隆抗体酶,在此基础上又应用基因工程方法制备了模拟GPX的小分子单链抗体(scFv)酶。这些抗体酶虽然活性较高,但来源于鼠的免疫原性限制了它们的医学应用。本研究的主要目的就是制备具有一定GPX活力的人源单链抗体酶,并在细胞水平上研究其抗氧化作用。主要开展了以下工作: 1.合成抗原GSH-S-DNPBu 根据罗贵民小组提出的疏水修饰理论合成了谷胱甘肽(GSH)的类似物GSH-S-DNPBu,用核磁共振对合成的化合物进行了分析。 2.亲和筛选抗GSH-S-DNPBu的噬菌体单链抗体通过吸附-洗脱-扩增步骤,从噬菌体展示人源单链抗体库(human synthetic VH+VL scFv library)中亲和筛选抗GSH-S-DNPBu的噬菌体单链抗体。用“多克隆噬菌体ELISA”监测筛选过程并用“单克隆噬菌体ELISA”选择可以和GSH-S-DNPBu特异性结合的单克隆噬菌体。 3.表达和纯化人源单链抗体根据“单克隆噬菌体ELISA”结果,提取选择的单克隆噬菌体DNA,应用PCR方法从噬菌体DNA中扩增出编码人源单链抗体的基因,将其克隆到分泌性表达载体pPELB中并转化大肠杆菌Rosetta,诱导转化菌Rosetta表达人源单链抗体。用Ni2+螯合亲和层析(IMAC)纯化可溶性表达的人源单链抗体。应用SDS-PAGE和western blot实验鉴定表达和纯化的人源单链抗体。 4.制备人源含硒单链抗体酶用化学突变法将人源单链抗体中的丝氨酸转变为硒代半胱氨酸(Sec),得到具有GPX活力的人源含硒单链抗体酶,其活力为1288 U/μmol。 5.在细胞水平上研究人源含硒单链抗体酶的抗氧化作用利用H2O2损伤大鼠乳鼠的心肌细胞建立氧化应激损伤模型,应用MTT法,丙二醛(MDA)测定试剂盒,乳酸脱氢酶(LDH-L)比色法试剂盒,流式细胞仪和活性氧测定试剂盒检测受损细胞的存活率、脂质过氧化程度、心肌细胞膜完整性、线粒体膜电位和细胞内ROS水平等指标的变化以及人源含硒单链抗体酶对上述指标的影响。结果表明人源含硒单链抗体酶能部分的增加心肌细胞存活率,减少脂质过氧化产物MDA生成,降低由于细胞膜完整性受损而引起的LDH泄漏,恢复线粒体膜电位,降低细胞内ROS含量,表明人源含硒单链抗体酶具有很强的抗氧化能力和良好的药用前景。
[Abstract]:Se-containing glutathione peroxidase (GPX) is the most important antioxidant enzyme in the body. There are many artificial mimic enzymes of GPX, but the activity is generally low. A thorough study of the existing GPX mimics showed that the production of substrate binding sites was one of the key factors for the preparation of highly active GPX mimics. The preparation of monoclonal antibody (McAb) is an effective method to produce substrate-binding receptor. In the process of preparing mimic GPX antibody enzyme, Luo Guimin team proposed hydrophobic modification theory and used the theory to prepare a series of monoclonal antibody enzymes with high GPX activity. On the basis of this, the small molecular single chain antibody (scFv) enzyme mimicking GPX was prepared by genetic engineering method. Although the activity of these antibody enzymes is high, the immunogenicity of these antibodies from mice limits their medical application. The main purpose of this study was to prepare human scFv with GPX activity and to study its antioxidant activity at cellular level. The main work is as follows: 1. The synthesis of glutathione (GSH) analogues based on hydrophobic modification theory proposed by Luo Guimin's group was studied by synthetic antigen GSH-S-DNPBu. The synthesized compounds were analyzed by nuclear magnetic resonance (NMR). 2. The phage scFv against GSH-S-DNPBu was screened by affinity screening by adsorption-elution amplification step. The phage scFv against GSH-S-DNPBu was screened by affinity from phage display human scFv library (human synthetic VH VL scFv library). The screening process was monitored by "polyclonal phage ELISA" and "monoclonal phage ELISA" was used to select the monoclonal phage that could specifically bind to GSH-S-DNPBu. 3. Expression and purification of human single-chain antibody based on the results of "Monoclonal phage ELISA", the selected monoclonal phage DNA, was used to amplify the gene encoding human single-chain antibody from phage DNA by PCR method. It was cloned into secretory expression vector pPELB and transformed into Escherichia coli Rosetta, to induce Rosetta to express human scFv. Soluble human scFv was purified by Ni2 chelating affinity chromatography (IMAC). The expressed and purified human scFv was identified by SDS-PAGE and western blot experiments. 4. Preparation of human Se-containing scFv enzyme conversion of serine from human scFv to selenocysteine (Sec), by chemical mutation method to obtain a human Se-containing scFv with GPX activity, the activity of which is 1288 U / 渭 mol. 5. Study on the Antioxidant effect of Human Se-containing single chain Antibody enzyme at the Cell level the oxidative stress injury model of neonatal rat cardiomyocytes damaged by H2O2 was established. The MTT assay and malondialdehyde (MDA) assay kit were used to detect the oxidative stress. Lactate dehydrogenase (LDH-L) colorimetric kit, flow cytometry and reactive oxygen species assay kit were used to detect the survival rate, lipid peroxidation and integrity of myocardial cell membrane. The changes of mitochondrial membrane potential and intracellular ROS level and the effect of human selenium single chain antibody enzyme on these indexes. The results showed that human scFv could partially increase the survival rate of myocardial cells, reduce the production of lipid peroxidation product MDA, reduce the leakage of LDH caused by the damage of cell membrane integrity, and restore mitochondrial membrane potential. The decrease of ROS content in cells indicates that human scFv has strong antioxidant ability and good medicinal prospect.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R341

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