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.     

Improving GPX activity of selenium‐containing human single‐chain Fv antibody by site‐directed mutation based on the structural analysis

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se‐scFv‐B3 (selenium‐containing single‐chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv‐B3 was carried out. A three‐dimensional (3D) structure of scFv‐B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer‐aided docking and energy minimization (EM) calculations of the antibody‐GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni²⁺‐immobilized metal affinity chromatography (IMAC), then transformed to selenium‐containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se‐scFv‐B3‐A180S was significantly increased compared to the original Se‐scFv‐B3. Copyright © 2009 John Wiley & Sons, Ltd.     

亲和层析法用于噬菌体抗体库的筛选

本研究报道一种基于固定化金属亲和层析(IMAC)的噬菌体抗体库液相筛选方法。将纯化的带有His标签的抗原与噬菌体抗体库混合,噬菌体抗体与抗原充分结合后再加入亲和介质,使噬菌体抗体抗原复合物通过His标签与介质结合,然后通过充分洗涤去除非特异性噬菌体抗体,最后将特异性噬菌体抗体洗脱下来,感染TG1,进行下一轮筛选。整个筛选过程中抗原与抗体的结合在液相中完成,不仅消除了固相介质对抗原表位的影响,也更有利于噬菌体抗体与抗原的充分作用。将此方法应用于HEV NE2蛋白特异性人源噬菌体抗体的筛选,抗原竞争ELISA,阳性血清阻断,可溶性单链抗体表达检测及测序结果表明,最终获得2个特异性人源抗体。     

人转铁蛋白受体及其特异性抗体的制备

目的:从胎盘中提取转铁蛋白受体并获得抗转铁蛋白受体的抗体。方法:人新鲜胎盘组织被破碎后,用去污剂TritonX-100裂解细胞膜,释放膜蛋白。利用膜蛋白中的转铁蛋白受体能与铁-转铁蛋白复合物特异性结合的特性对其进行亲和纯化。对纯化得到的目的蛋白,经脱盐后进行ELISA及肽质量图谱分析,证明为所需的转铁蛋白受体后,以其包被免疫管,从全合成人源噬菌体抗体库中筛选抗体。结果:从人源噬菌体抗体库中筛选到5个能够与转铁蛋白受体特异性结合的噬菌体单链抗体。结论:以人源转铁蛋白受体为抗体,可从全人源噬菌体抗体库中筛选到其特异性的抗体。     

从噬菌体抗体库中淘选血纤维蛋白特异的单链抗体

为构建小鼠噬菌体抗体库 ,以获得对人血纤维蛋白特异的抗体 ,由小鼠脾脏提取 m RNA,经反转录 PCR扩增出抗体重链、轻链可变区基因片段 ,将二者和一段编码十五肽 (Gly4 Ser) 3的 DNA接头借助重组 PCR组装成为单链抗体 (single- chain antibody,Sc Ab)基因 .将单链抗体基因插入噬菌体展示载体 p CANTAB- 5E,通过电击法转化大肠杆菌 TG1细胞 ,用辅助噬菌体 M1 3K0 7超感染 ,构建了库容量在 1 0 8以上的噬菌体单链抗体库 .利用亲和选择方法 (淘选 ) ,从噬菌体抗体库中选得血纤维蛋白特异的单链抗体 .模拟抗体成熟过程 ,用 DNA改组 (DNA shuffling)技术使抗体基因重新组合 ,构建新的改组抗体库 ,并从中选择到提高了亲和力的噬菌体单链抗体 .抗体基因在大肠杆菌中表达 ,表达蛋白经 Sephadex G- 75柱层析分离 ,得到初步纯化的单链抗体蛋白 .     

Construction of human Fab (gamma1/kappa) library and identification of human monoclonal Fab possessing neutralizing potency against Japanese encephalitis virus

A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from Japanese encephalitis virus hyper-immune volunteers on pComb3H phagemid vector. The size of the constructed Fab library was 3.3x10(8) Escherichia coli transformants. The library was panned 3 times on the purified Japanese encephalitis virus (JEV) virion, and phage clones displaying JEV antigen-specific Fab were enriched. The enriched phage pool was then screened for clones producing Fab molecule with JEV neutralizing activity by the focus reduction-neutralizing test. Among 188 randomly selected clones, 9 Fab preparations revealed neutralizing activities against JEV strain Nakayama. An E. coli transformed with TJE12B02 clone, which produced human monoclonal Fab with the highest neutralizing activity was cultured in a large scale, and the Fab molecule was purified using affinity chromatography. The purified FabTJE12B02 showed the 50% focus reduction endpoint at the concentration of 50.2 microg/ml (ca. 1,000 nM) when JEV strain Nakayama was used. The FabTJE12B02 recognized E protein of JEV strain Nakayama, and the dissociation equilibrium constant (Kd) of the FabTJE12B02 against purified JEV antigen was calculated as 1.21x10(-8) M. Sequence analysis demonstrated that TJE12B02 used a VH sequence homologous to the VH3 family showing 88.8% homology to germline VH3-23, and used a Vkappa sequence homologous to the VkappaII subgroup showing 92.8% homology to germline A17.     

人源中和性抗汉滩病毒单克隆抗体Fab段基因的获得和表达

运用噬菌体表面表达技术,获得人源和中性抗滩滩病毒汉滩型Ｇ１基因工程单克隆抗体Ｆａｂ段基因及其表达,并同时获得抗汉滩病毒核蛋白的Ｆａｂ抗体。从能综合征出血热疫区恢复期病人抗凝血中分离到的外周淋巴细胞中,提取了部细胞ＲＮＡ。通过ＲＴ－ＰＣＲ方法,用一组人ＩｇＧ Ｆａｂ基因特异性引物,从合成了ｃＤＮＡ中经ＰＣＲ扩增了一组轻链和重链Ｆａｂ段基因,将轻链和重链先后插入噬菌体载体ｐＣｏｍｂ３,ｄｎａｌｆ ｖｆ     

Single-chain antibody against human lipocalin-type prostaglandin D synthase: Construction, expression, purification, and activity assay

An active form of single-chain antibody (ScFv) from murine monoclonal antibody 4A7, which is specific for lipocalin-type prostaglandin D synthase (L-PGDS), was produced in Escherichia coli. The complementary DNA fragments encoding the variable regions of heavy chain (VH) and light chain (VL), which amplified from hybridoma 4A7 producing a monoclonal antibody (IgG1) against L-PGDS, were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The resultant ScFv were cloned into the vector pGEM and expressed in E. coli as inclusion bodies. The expressed ScFv fusion proteins were purified by Ni2+-nitrilotriacetic acid chromatography. The purity and activity of purified ScFv were confirmed by SDS-PAGE and ELISA. The result revealed that 4A7 ScFv conserved the same characteristics of specific recognition and binding to sperm as the parental 4A7 monoclonal antibody.     

一种新的乙型肝炎病毒表面抗原结合蛋白的筛选、表达及其生物学活性的初步研究

为了研究乙肝病毒侵染肝细胞过程中的功能蛋白 ,通过印迹免疫分析技术从人肝cDNA噬菌体表达库中筛选出一株编码乙肝表面抗原结合蛋白 (hepatitisBsurfaceantigenbindingprotein ,HBsAg BP)的cDNA克隆 .基因测序结果表明 ,该cDNA具有独立的开放阅读框架 ,编码 1个由 344个氨基酸残基构成的可溶性蛋白分子 ,属于免疫球蛋白超家族成员 .将该基因克隆到原核表达载体pTriplEx后 ,在E .coliXL1 Blue菌株中获得 4 4kD的重组蛋白 .重组蛋白经Western印迹和ELISA实验证明具有与乙肝表面抗原特异性结合的能力 .进一步经流式细胞仪实验显示 ,在纯化的重组蛋白存在的情况下 ,天然的HBsAg与肝细胞株HepG2的亲和力显著增高 .结果显示 ,该乙肝表面抗原结合蛋白可能是介导乙肝病毒对肝细胞亲和侵染的可溶性辅助受体 .     

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.     

蛋白酪氨酸磷酸酶PTP1B催化活性区的原核表达及活性分析

从GenBank获得人PTP1B催化活性区(PTP1Bc)氨基酸序列(1~301aa), 通过重叠PCR获得PTP1Bc基因。构建 pET-22b(+)/PTP1Bc原核表达载体, 转化大肠杆菌BL21(DE3), 阳性重组子IPTG诱导表达, Ni柱纯化蛋白。目的蛋白以包涵体的形式表达, 表达量占菌体总蛋白30%以上。纯化后, 蛋白纯度达95%以上。Western blotting结果表明所得的蛋白可与抗 PTP1B抗体发生特异性结合; 酶活实验证实复性的蛋白具有一定的磷酸酶活性。PTP1Bc基因的构建、表达纯化及活性分析, 为进一步的功能研究奠定了基础。     

蛋白酪氨酸磷酸酶PTP1B催化活性区的原核表达及活性分析

从GenBank获得人PTP1B催化活性区(PTP1Bc)氨基酸序列(1~301aa), 通过重叠PCR获得PTP1Bc基因。构建 pET-22b(+)/PTP1Bc原核表达载体, 转化大肠杆菌BL21(DE3), 阳性重组子IPTG诱导表达, Ni柱纯化蛋白。目的蛋白以包涵体的形式表达, 表达量占菌体总蛋白30%以上。纯化后, 蛋白纯度达95%以上。Western blotting结果表明所得的蛋白可与抗 PTP1B抗体发生特异性结合; 酶活实验证实复性的蛋白具有一定的磷酸酶活性。PTP1Bc基因的构建、表达纯化及活性分析, 为进一步的功能研究奠定了基础。     

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。     

Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions

A structure-based approach was used to design libraries of synthetic heavy chain complementarity determining regions (CDRs). The CDR libraries were displayed as either monovalent or bivalent single-chain variable fragments (scFvs) with a single heavy chain variable domain scaffold and a fixed light chain variable domain. Using the structure of a parent antibody as a guide, we restricted library diversity to CDR positions with significant exposure to solvent. We introduced diversity with tailored degenerate codons that ideally only encoded for amino acids commonly observed in natural antibody CDRs. With these design principles, we reasoned that we would produce libraries of diverse solvent-exposed surfaces displayed on stable scaffolds with minimal structural perturbations. The libraries were sorted against a panel of proteins and yielded multiple unique binding clones against all six antigens tested. The bivalent library yielded numerous unique sequences, while the monovalent library yielded fewer unique clones. Selected scFvs were converted to the Fab format, and the purified Fab proteins retained high affinity for antigen. The results support the view that synthetic heavy chain diversity alone may be sufficient for the generation of high-affinity antibodies from phage-displayed libraries; thus, it may be possible to dispense with the light chain altogether, as is the case in natural camelid immunoglobulins.     

化学修饰抗体增加模拟酶的GPX活性

将丝氨酸共价偶联到兔抗人ＩｇＭ（Ｆｃμ）抗体上,以增加抗体结合部位丝氨酸残基含量．在中性条件下,丝氨酸被对苯甲基磺酰氟活化,再经ＮａＨＳｅ亲核取代,将丝氨酸诱变为硒代半胱氨酸（ＳｅＣｙｓ）．其谷胱甘肽过氧化物酶（ＧＰＸ）活性由未增加丝氨酸前的诱变抗体的７０Ｕ／μｍｏｌ提高到２１８Ｕ／μｍｏｌ,其活性为模拟物ＰＺ５１的２００多倍．     

Identification of a human monoclonal Fab with neutralizing activity against H3N2 influenza A strain from a newly constructed human Fab library

A combinatorial Fab library was constructed in pComb3H phagemid vectors, using RNA from peripheral blood lymphocytes of a healthy volunteer who had recovered from an influenza A virus infection. The library contained approximately 1.3 x 10(8)E. coli transformants. Bio-panning was carried out against an influenza vaccine containing components of influenza A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), and B/Shandong/7/97 for the enrichment of phages displaying human Fab specific to the viral proteins. E. coli transformed with IF1A11, 1 of 94 randomly selected clones, displayed a human Fab antibody molecule (FabIF1A11) with efficient neutralizing activity against H3N2 influenza A virus strains. The purified FabIF1A11 demonstrated neutralizing activity against A/Okayama/6/01 (H3N2) and A/Kitakyushu/159/93 (H3N2) with 50% plaque reduction neutralization titers of 0.11 microg/ml (2.2 nM) and 1.4 microg/ml (28 nM) respectively. However, FabIF1A11 did not show neutralizing activity against the influenza A virus strain A/USSR/77 (H1N1) or the influenza B virus strain B/Kanagawa/73, even at a concentration of 20 microg/ml (400 nM). The Kd of FabIF1A11 was calculated as 3.6 x 10(-9) M. FabIF1A11 was estimated to recognize a conformational epitope on the hemagglutinin of A/Okayama/6/01 (H3N2). The human monoclonal Fab product FabIF1A11 may have potential as a therapeutic or short-term prophylactic molecule for humans with influenza A H3N2 infection.     

利用B细胞培养和RT-PCR技术从我国HIV-1感染者中筛选膜蛋白特异性单克隆抗体的初步研究

本研究通过采集1型人类免疫缺陷病毒(Human immunodeficiency virus-1,HIV-1)感染者抗凝全血,分离出外周血单个核细胞,然后用磁珠分选纯化记忆性B细胞和体外活化记忆性B细胞,促使其分泌抗体,用ELISA法识别阳性B细胞克隆,并提取阳性B细胞的RNA,从中扩增抗体重链和轻链基因并克隆到表达载体中,再用携带重链基因的质粒和携带轻链基因的质粒共转染293T细胞,获得HIV-1特异性人单克隆抗体,进行抗体特性的鉴定。结果从1例HIV-1感染者的记忆性B细胞中筛选出了4株HIV-1包膜糖蛋白(Envelope glycoprotein,Env)特异性人单克隆抗体,其中2株具有较好的抗体依赖细胞介导的细胞毒作用活性,另有1株对HIV-1假病毒有较弱的中和活性。说明我们成功地引进了利用B细胞培养和RT-PCR技术从人体淋巴细胞中筛选特异性抗体基因的人单克隆抗体技术平台。用该技术可以成功获得HIV-1Env特异性单克隆抗体,为将来从能产生高滴度广谱中和抗体的感染者体内筛选广谱中和抗体打下了基础。     

人源天然ScFv噬菌体抗体库的构建及鉴定

噬菌体抗体库技术是获得治疗性抗体的一条重要途径。以20份健康人外周血为样本,通过提取淋巴细胞、逆转录－PCR（RT PCR）、抗体可变区基因的扩增、重叠PCR获得单链抗体（ScFv）基因,将ScFv克隆入噬粒载体,通过近300次的电转化获得了库容量为1.3×109的全人源天然ScFv噬菌体抗体库。通过随机挑克隆测序和用5种不同抗原筛选对抗体库进行了初步验证。随机测序表明抗体库具有较好的多样性,用5种不同抗原对其进行筛选,均获得了特异性噬菌体抗体的不同富集,表明成功构建了一个多样性良好的人源天然ScFv噬菌体抗体库。     

RNA recognition by a human antibody against brain cytoplasmic 200 RNA

Diverse functional RNAs participate in a wide range of cellular processes. The RNA structure is critical for function, either on its own or as a complex form with proteins and other ligands. Therefore, analysis of the RNA conformation in cells is essential for understanding their functional mechanisms. However, no appropriate methods have been established as yet. Here, we developed an efficient strategy for panning and affinity maturation of anti-RNA human monoclonal antibodies from a naïve antigen binding fragment (Fab) combinatorial phage library. Brain cytoplasmic 200 (BC200) RNA, which is also highly expressed in some tumors, was used as an RNA antigen. We identified MabBC200-A3 as the optimal binding antibody. Mutagenesis and SELEX experiments showed that the antibody recognized a domain of BC200 in a structure- and sequence-dependent manner. Various breast cancer cell lines were further examined for BC200 RNA expression using conventional hybridization and immunoanalysis with MabBC200-A3 to see whether the antibody specifically recognizes BC200 RNA among the total purified RNAs. The amounts of antibody-recognizable BC200 RNA were consistent with hybridization signals among the cell lines. Furthermore, the antibody was able to discriminate BC200 RNA from other RNAs, supporting the utility of this antibody as a specific RNA structure-recognizing probe. Intriguingly, however, when permeabilized cells were subjected to immunoanalysis instead of purified total RNA, the amount of antibody-recognizable RNA was not correlated with the cellular level of BC200 RNA, indicating that BC200 RNA exists as two distinct forms (antibody-recognizable and nonrecognizable) in breast cancer cells and that their distribution depends on the cell type. Our results clearly demonstrate that anti-RNA antibodies provide an effective novel tool for detecting and analyzing RNA conformation.