催化性抗体及其研究进展

根据过渡态理论,按特定的化学反应机制确定反应中的可能过渡态结构,选择和该过渡态结构类似的化合物作为半抗原,可诱导机体产生具有催化活性的催化性抗体.文章对诱导催化性抗体中半抗原的选择原则、催化性抗体和非催化性抗体间的联系、催化性抗体和酶促催化反应的比较等方面进行了较为全面的综述,并对催化性抗体在医药科学中的应用前景及限制因素进行了讨论.     

油包水微乳液中抗体酶催化布洛芬酯选择性水解的酶学特性

根据过渡态理论设计和合成了能诱导产生催化选择性水解布洛芬甲酯的催化抗体的四面体硫酸盐半抗原,并与牛血清白蛋白(BSA)偶联制备成免疫源,通过免疫手段成功筛选出具有加速选择性水解生成S-布洛芬的特异性催化抗体.其Kcat,app/Kuncat,app达1.6x104.进一步地将催化抗体运用到W/O微乳体系(反胶束)中进行布洛芬酯的选择性水解研究,其动力学研究证明其催化过程同样遵循Michaelis.Menten方程.考察了pH值和温度对催化初速度影响,Wo(体系中水和琥珀酸二辛酯磺酸钠(AOT)的摩尔比)对催化初速度影响呈现为钟罩型,最适的Wo.为21.     

内抗体——一种新的基因功能研究工具

细胞内抗体是指能够在细胞内特定区域特异性中和靶蛋白功能的抗体.内抗体技术是一种翻译后水平上抑制靶蛋白质功能的技术,已经成为一种功能基因组学研究的重要工具.本文综述了细胞内抗体转运和作用机制,分析了细胞内抗体技术和其他靶向敲除技术的优、缺点,以及细胞内抗体技术存在的问题及解决途径.     

OGT 多克隆抗体的制备及特异性分析

目的:为了探讨O-GlcNAc糖基转移酶OGT的生理和病理作用,需制备能高效特异性检测OGT的抗体。方法:在NCBI数据库中,查找人源OGT基因序列,根据OGT的结构特点,选取OGT的C末端催化结构域中的一段多肽序列(464-949位点氨基酸)做抗原。首先,构建OGT的C末端催化结构域(464-949位点氨基酸)的重组表达载体pET30-a-OGT-C,转化至大肠杆菌BL21(DE3)感受态细胞中,IPTG诱导表达融合His标签的OGT-C蛋白,Ni+珠亲和层析法纯化提取OGT-C蛋白。再以OGT-C重组蛋白作为抗原,免疫Wistar大鼠制备多克隆抗体,并用间接ELISA法检测OGT抗体的效价,Western blotting鉴定抗体特异性。结果:多抗效价达1:80000;在免疫印迹实验中,此多抗可以高效的检测重组抗原,并可以特异性识别培养细胞内源表达的ncOGT和mOGT这2种OGT亚型。结论:实验结果表明,获得高效价、高特异性的OGT多克隆抗体,在OGT的生物学研究中可以用于检测ncOGT和mOGT的表达。     

提高抗体酶催化活性的研究进展

提高抗体酶催化活性的研究进展王俊＊梁世中（华南理工大学生物工程系,广州５１０６４１）＊现工作单位：广州暨南大学生物工程学系（５１０６３２）。关键词抗体酶催化抗体催化活性抗体酶（催化抗体）技术是化学和生物学的研究成果在分子水平交叉渗透的产物,是将抗体的...     

生物催化剂研发及生物催化技术的产业化

资源危机与环境压力已经成为现代人类社会实现可持续发展的主要瓶颈,着眼于发展环境友好、过程高效的工业生物技术,有望对社会发展产生巨大的引领和带动作用,工业生物技术的发展将成为解决能源、环境和资源问题的关键,而生物催化是工业生物技术的核心技术.本文介绍了生物催化在工业可持续发展中的地位,国内外研究进展及其影响,并对新型生物催化剂的发现与新的催化功能的开发应用实例进行了介绍.     

生长抑素抑制剂—半胱胺促进大鼠,幼兔生长的研究

近年来许多研究表明,注射生长抑素(Somatostatin,SS)产生的抗体可消除SS的作用.加速幼畜的生长。但由于此法需制备高效价的抗体,或以SS纯品主动免疫动物以及存在机体对抗原、抗体的清除率等问题,因而在应用于生产之前,还需进一步深入研究。1981年Szabo等报道口服半胱     

改造大肠杆菌合成海藻糖途径以高效合成海藻糖

海藻糖是相容性溶质的一种,因其具有多种生物学功能,在食品、化妆品、药品以及器官移植等方面均有很广泛应用。然而近几年生产海藻糖主要集中在使用酶催化的方法,虽然这种方法的转化效率高,但是却存在着副产物的问题,难以得到高纯度的海藻糖产品,严重制约了海藻糖的应用。本文通过基因工程技术在大肠杆菌Escherichia coli中构建了海藻糖高效合成新途径,通过全细胞催化合成海藻糖。利用PCR技术在哈氏噬纤维菌Cytophaga hutchinsonii中克隆获得海藻糖双功能合成酶基因(tpsp),采用E.coli pTac-HisA高效表达载体,实现海藻糖双功能合成酶基因(tpsp)高效表达,利用高效表达菌株进行全细胞催化,将葡萄糖高效转化为海藻糖。结果表明C.hutchinsonii海藻糖合成酶基因(tpsp)在E.coli中成功实现表达,该酶能够在胞内将葡萄糖高效转化为海藻糖,并将其转运到胞外,实现海藻糖的高效率合成,海藻糖的产量提高到1.2 g/L,相对转化率为21%。当将此高产菌株在发酵罐中进行转化时,海藻糖的产量达到13.3 g/L,葡萄糖的相对转化率达到48.6%。采用C.hutchinsonii海藻糖合成酶基因高效表达并且应用于海藻糖全细胞合成催化在国内外尚属首次报道,海藻糖的转化率及产率都已达到文献报道最高水平,本研究为开拓海藻糖生产新技术奠定了基础。     

植物表达抗体的研究与发展

利用植物表达抗体是近年来兴起的植物基因工程的一个新领域.它将编码抗体或抗体片段的基因导入植物,从而在植物中产生全长抗体或抗体片段.利用植物表达抗体最诱人的潜在用途是可以大规模廉价生产治疗和诊断用抗体.此外,植物抗体还能够通过调控植物代谢改良植物性状并赋予植物对病虫害的抗性.目前植物抗体的商品化还存在一些问题.     

抗体分离纯化技术的研究进展

制备高特异性、高效价的抗体是实验免疫学技术的基础,抗体质量的高低,将直接影响试验的成败.抗体的制备有两个途径:一是一般通用的方法,以纯化的抗原免疫动物,获得多克隆抗体;二是应用杂交瘤技术制备单克隆抗体.但不论用何种技术制备的抗体都需要进行纯化.重要的是根据抗体的性质和来源选择一个合适的分离纯化方法.对当前的纯化方法进行了一个简要的综述.     

Overpassing an aberrant V(kappa) gene to sequence an anti-idiotypic abzyme with (beta)-lactamase-like activity that could have a linkage with autoimmune diseases.

A monoclonal antibody 9G4H9 that exhibits a beta-lactamase-like activity was previously obtained in accordance with the idiotypic network theory. This abzyme presents the most catalytic efficiency in amidase activity described in literature (kcat = 0.9 min-1). Some reports have demonstrated that functionality as complex as catalysis may be mimicked in this way. Comparison of the catalytic properties of both enzyme and abzyme previously allowed us to obtain better knowledge about 9G4H9 abzymatic machinery. In attempt to characterize this abzyme, the variable regions of kappa and heavy chain were cloned. We present a 'universal' method to clone the correct Vkappa gene to bypass aberrant Vkappa (abVkappa) produced by MOPC-21-derived hybridomas. Sequences obtained are compared in the GenBank database. The VH and Vkappa genes present some important sequence homology with autoantibodies suggesting a direct relationship between catalytic anti-idiotypic antibody and autoimmunity.     

Studies on the catalytic behaviour of a cholinesterase-like abzyme in an AOT microemulsion system

The hydrolytic activity of a monoclonal catalytic antibody (9A8) (abzyme) with acetylcholinesterase-like activity was investigated in water-in-oil (w/o) microemulsions (reverse micelles) based on sodium bis-2-(ethylhexyl)sulfosuccinate (AOT) in isooctane, using p- and o-nitrophenylacetate (p-and o-NPA) as substrates. The dependence of the abzyme hydrolytic activity on the molar ratio of water to surfactant (w(o)) showed a bell-shaped curve, presenting a maximum at w(o)=11.1. An increase of the AOT concentration at constant w(o), resulted in a decrease of the catalytic activity suggesting a possible inhibition effect of the surfactant. The incorporation of the abzyme into the reverse micelle system caused a blue shift of the fluorescence emission maximum by a magnitude of 7-10 nm depending on the w(o) value. This result indicates that the antibody molecule, or a large part of it, is located in the aqueous microphase of the system. Kinetic studies showed that the hydrolysis of p-and o-NPA in microemulsion system as well as in aqueous solution follows Michaelis-Menten kinetics. The catalytic efficiency (k(cat)/K(m)) in w/o microemulsion was significant lower than in aqueous solution.     

A new human catalytic antibody Se‐scFv‐2D8 and its selenium‐containing single domains with high GPX activity

Glutathione peroxidase (GPX) is a well‐known antioxidant selenoenzyme, which can catalyze the reduction of a variety of hydroperoxides and consequently protect cells and other biological tissues against oxidative damage. Many attempts have been made to mimic its function, and a human catalytic antibody Se‐scFv‐B3 with GPX activity has been prepared in our previous study. This time, a new clone 2D8 that bound specifically to the glutathione analog GSH‐S‐DNPBu was selected again by using the technology of phage display antibody library, and then scFv‐2D8 was successfully expressed in soluble form and purified using Ni²⁺‐immobilized metal affinity chromatography. After being converted into selenium‐containing scFv by chemically modification, it showed higher GPX activity than previous abzyme Se‐scFv‐B3. The heavy chain variable fragment of scFv‐2D8 was also prepared and converted into selenium‐containing protein using the same method. This selenium‐containing single‐domain antibody showed some GPX activity and, to the best of our knowledge, is the first human single‐domain abzyme with GPX activity, which lays a foundation for preparing GPX abzyme with human origin, lower molecular weight and higher activity. Copyright © 2009 John Wiley & Sons, Ltd.     

Molecular simulation studies of a selenium-containing scFv catalytic antibody that mimics glutathione peroxidase

GPX is a mammalian antioxidant selenoenzyme which protects biomembranes and other cellular components from oxidative damage by catalyzing the reduction of a variety of hydroperoxides (ROOH), using Glutathione (GSH) as the reducing substrate. The single-chain Fv fragment of the monoclonal antibody 2F3 (scFv2F3) can be converted into the selenium-containing Se-scFv2F3 by chemical modification of the serine. The new selenium-containing catalytic antibody Se-scFv2F3 acts as a glutathione peroxidase (GPX) mimic with high catalytic efficiency.In order to investigate which residue of scFv2F3 is converted into selenocysteine and to describe the proper reaction site of GSH to Se-scFv2F3, a three-dimensional structure of scFv2F3 is built by means of homology modeling. The 3D model is assessed by molecular dynamics (MD) simulation to determine its stability and by comparison with those of known protein structures. After the serine in the scFv2F3 is modified to selenocysteine, a catalytic antibody (abzyme) is obtained. From geometrical considerations, the solvent-accessible surface of the protein is examined. The computer-aided docking and energy minimization (EM) calculations of the abzyme–GSH complex are then carried out to explore the possible active site of the glutathione peroxidase mimic Se-scFv2F3. The structural information from the theoretically modeled complex can help us to further understand the catalytic mechanism of GPX.     

Dynamics of antibody nuclease activity in blood of women during pregnancy and lactation

In human milk we previously found catalytic antibodies (abzymes) catalyzing hydrolysis of DNA, RNA, NMP, NDP, and NTP and also phosphorylation of proteins and lipids. In the present study we have analyzed nuclease activities of antibodies in blood of women during pregnancy and lactation. Blood of healthy male and female volunteers lacked catalytically active antibodies, whereas antibodies from blood of pregnant women hydrolyzed DNA and RNA and their relative activity varied over a wide range. Relative blood abzyme activities significantly increased after delivery and at the beginning of lactation. The highest abzyme activity was observed in blood of parturient women. Although the dynamics of changes in antibody DNase activity during pregnancy was rather individual for each woman, there was a common trend in the increase in antibody activity in the first and/or third trimester of the pregnancy. The DNase activity of IgG and IgM from blood of healthy pregnant women was 4-5 times less than that from pregnant women with pronounced autoimmune thyroiditis.     

Peculiarities of abzymes from sera and milk of healthy donors and patients with autoimmune and viral diseases

The detection of catalytic activity of antibodies is the earliest indicator of development of autoimmune diseases (AID). In early stages of AID, the repertoire of abzymes with various properties is relatively small, but it is greatly increased during their development. Catalytic diversity of the abzymes includes DNase, RNase, ATPase, and oxidoreductase activities; there are antibodies phosphorylating proteins, lipids, and polysaccharides. This review summarizes new data on abzyme heterogeneity and possible reasons for this phenomenon. A possible role of abzymes and their exceptional multiplicity in the pathogenesis of different AID is discussed.     

Creation of catalytic antibodies metabolizing organophosphate compounds

Development of new ways of creating catalytic antibodies possessing defined substrate specificity towards artificial substrates has important fundamental and practical aspects. Low immunogenicity combined with high stability of immunoglobulins in the blood stream makes abzymes potent remedies. A good example is the cocaine-hydrolyzing antibody that has successfully passed clinical trials. Creation of an effective antidote against organophosphate compounds, which are very toxic substances, is a very realistic goal. The most promising antidotes are based on cholinesterases. These antidotes are now expensive, and their production methods are inefficient. Recombinant antibodies are widely applied in clinics and have some advantage compared to enzymatic drugs. A new potential abzyme antidote will combine effective catalysis comparable to enzymes with high stability and the ability to switch on effector mechanisms specific for antibodies. Examples of abzymes metabolizing organophosphate substrates are discussed in this review.     

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。     

Reverse micelles,a system for antibody-catalysed reactions

This work presents some aspects of the application of catalytic antibodies in water-in-oil microemulsions (reverse micelles) based on sodium bis-2-(ethylhexyl)sulfosuccinate (AOT) in isooctane. The monoclonal antibody (mAb) 9A8 used in this study is a fully characterised acetylcholinesterase-like antibody produced by the anti-idiotypic approach. The effect of various parameters, such as, the size and the concentration of reverse micelles, as well as the concentration and the nature of substrates on abzyme catalytic activity were investigated.     

Formation of different abzymes in autoimmune-prone MRL-lpr/lpr mice is associated with changes in colony formation of haematopoietic progenitors

It was shown that IgGs from the sera of 2-7-month-old control non-autoimmune (CBA x C57BL)F1 and BALB/c mice and 2-3-month-old autoimmune prone MRL-lpr/lpr mice (conditionally healthy mice) are catalytically inactive. During spontaneous development of deep systemic lupus erythematosus (SLE)-like pathology a specific reorganization of immune system of these mice leads to conditions associated with a production of IgGs hydrolyzing DNA, ATP and polysaccharides with low catalytic activities (conditionally pre-diseased mice).A significant increase in DNase, ATPase and amylase IgG relative activities associated with a transition from pre-diseased to deep diseased mice is correlated with additional changes in differentiation and proliferation of mice bone marrow haematopoietic stem cells (HSCs) and lymphocyte proliferation in different organs.The highest increase in all abzyme activities was found in mice immunized with DNA, which in comparison with pre-diseased and diseased mice are characterized by a different profile of HSC differentiation and by a suppression of cell apoptosis. Abzyme activities in the serum of pregnant females were comparable with those for pre-diseased mice, but the profile of HSC differentiation and cell apoptosis levels in pregnant and pre-diseased mice were quite different. Right after the beginning of lactation (4 days after delivery) and in a late time of lactation (14 days after delivery) there was an observed increase in cell apoptosis and two different stages of significant change in the HSC differentiation profiles; the first stage was accompanied with a significant increase and the second with a remarkable decrease in abzyme activities. Overall, all mouse groups investigated are characterized by a specific relationship between abzyme activities, HSC differentiation profiles, levels of lymphocyte proliferation, and cell apoptosis in different organs. From our point of view, the appearance of ATPase, DNase activities may be considered the earliest statistically significant marker of mouse spontaneous SLE and a further significant increase in their activities correlates with the appearance of SLE visible markers and with an increase in concentrations of anti-DNA Abs and urine protein. However, development of autoimmune (AI)-reactions and the increase in the sera anti-DNA antibodies (Abs) and in the abzyme activities in pregnant and lactating mice do not associate with SLE visible markers and proteinuria. The possible differences in immune system reorganizations during pre-disease, disease, pregnancy and lactation leading to production of different auto-antibodies and abzymes are discussed.