基因工程抗体融合蛋白的构建

抗体融合蛋白可以具有抗体的特性和所融合的功能蛋白的活性,可广泛用于免疫治疗、免疫诊断、抗体纯化及抗体和抗原的分析定量等,特别可用于免疫导向药物的制备。基因工程抗体融合蛋白比传统的化学交联的抗体融合蛋白具有更多的优越性。本文就基因工程抗体融合蛋白的构建和性质做一综述。     

人源抗严重急性呼吸综合征(SARS)病毒基因工程抗体的初步研究

严重急性呼吸道综合征(severe acute respiratory syndrome,SARS)或称传染性非典型肺炎,已严重威胁人民健康和生命安全。快速研制一种可用于紧急预防SARS病毒感染的基因工程抗体预防制剂迫在眉睫。为此,运用噬菌体表面呈现技术,从多个SARS病人恢复期血中获得淋巴细胞,通过基因工程手段,构建了人源抗SARS病毒基因工程抗体文库,并筛选获得37株特异抗SARS病毒基因工程Fab抗体,其中ll株人源抗体结合基因工程重组的SARS病毒核(N)蛋白,其中的1株在Western blot分析中与SARS病毒结合,识别SARS病毒N蛋白线性位点。对所获抗体的功能鉴定及基因分析正在进行中。人源抗SARS病毒基因工程抗体的获得,将对SARS疾病的特异性预防,治疗和诊断提供新的途径。     

基因工程小分子抗体的研究进展

随着分子生物学的发展,基因工程小分子抗体越来越受到人们关注,该抗体具有分子量小、结构简单、穿透性强、免疫源性低、低成本生产的优点,并且可与毒素、放射性核素、酶、细胞因子等结合用于肿瘤的诊断和治疗,临床应用前景十分广阔。对基因工程小分子抗体近几年的发展与应用作一综述。     

抗体库的构建与特异性抗体的筛选

噬菌体抗体库技术和出现标志着抗体技术从多克隆抗体,单克隆抗休进入基因工程抗体的时代,这一技术具有省时,省力,筛选容量大,可直接接得到抗体,便于构建筑各种基因工程抗体及在原核系统中进行表达的特点。     

Ataxin-3融合蛋白表达、纯化及抗血清的制备

目的:表达GST-ataxin-3-N融合蛋白并制备GST-ataxin-3特异性抗体,为深入研究其功能及其在SCA3发病机制中的作用提供重要的技术和材料保障.方法:将人ataxin-3氨基端基因克隆入原核表达载体pGEX-4T-2,在大肠杆菌(E.coli)BL21中表达,用Glutathione sepharose4B凝胶亲和柱纯化目的蛋白.利用纯化的GST-ataxin-3-N蛋白制备多克隆抗体.结果:成功构建了原核表达载体,得到高表达量的融合蛋白,经亲和层析柱纯化获得较高纯度的GST-ataxin-3-N融合蛋白.以融合蛋白免疫新西兰兔得到Ataxin-3-N多克隆抗体,Western Blotting及免疫荧光均证实该抗体能够识别Ataxin-3-myc蛋白,具有较高特异性.结论:利用原核表达人GST-ataxin-3-N融合蛋白制备的Ataxin-3多克隆抗体具有较好的特异性,可用于该蛋白的相关研究.     

人类新细胞因子CKLFSF2羧基端蛋白在大肠杆菌中的表达纯化、抗体制备与鉴定

哺乳动物细胞表达的人类新细胞因子——趋化素样因子超家族成员-2(CKLFSF2)存在分泌形式,位于CKLFSF2分子的羧基端,具有细胞趋化作用.为进一步研究CKLFSF2羧基端蛋白的结构和生物学功能及抗体制备,构建了GST-CKLFSF2C51原核表达质粒,经原核表达、亲和层析、凝胶过滤,获得GST-CKLFSF2C51融合蛋白和CKLFSF2羧基端蛋白(CKLFSF2C51),纯度可达到95%以上.GST-CKLFSF2C51融合蛋白用于制备多克隆抗体,ELISA方法检测抗体效价阳性,蛋白质印迹检测CKLFSF2哺乳动物细胞超表达细胞裂解液,获得特异性条带与预期大小一致.CKLFSF2C51经N端测序,质谱鉴定与预期结果一致,该蛋白质具有对PC-3细胞趋化的活性,并且该活性可被制备的多克隆抗体中和.上述结果表明,原核CKLFSF2羧基端蛋白具有与CKLFSF2真核表达蛋白类似的细胞趋化活性,原核CKLFSF2羧基端蛋白制备的多克隆抗体可用于免疫组织化学、蛋白质印迹检测,并能中和CKLFSF2蛋白的趋化活性作用.     

抗CD20单链抗体与力达霉素强化融合蛋白的制备及其抗肿瘤活性

抗体融合蛋白是新一代抗肿瘤抗体药物.CD20在约95%的B细胞非霍奇金淋巴瘤表面过度表达,是治疗B细胞淋巴瘤的理想靶点.力达霉素(LDM)是强效烯二炔抗肿瘤抗生素,目前已进入Ⅱ期临床阶段.采用DNA重组技术,利用大肠杆菌表达体系,制备抗CD20单链抗体与力达霉素辅基蛋白LDP的基因工程融合蛋白scFv-LDP.经纯化和...     

基因工程抗体

通过基因工程可以大规模地制备能与人相容的单克隆抗体或其片段,用于诊断、治疗以及抗体结构与功能的研究。基因工程抗体的制备过程是通过PCR技术获得抗体或其片段的基因,再与适当的载体重组后引入不同的宿主系统,如哺乳动物细胞、昆虫细胞、大肠杆菌和植物中进行表达、装配。较详细地介绍了基因工程抗体的背景、现状和进展。     

基因工程抗体的研究进展

基因工程抗体以其独特的优点（免疫原性低、可按人的意愿加以改造等）正逐渐取代动物源性单抗。随着基因工程和蛋白质工程等生物技术在抗体研制领域的广泛应用,适应不同需要的基因工程抗体的种类日趋多样化,构建日趋合理化,在体内的生物学效应与日臻完善,使之较天然单抗的治疗效果更好,范围更广,并在初步临床试用中展示了光辉的前景。     

HIV-1B′/C亚型Rev蛋白的表达、纯化与抗体制备

目的:为方便实验室工作中对HIV-1 B′/C亚型Rev蛋白的检测,制备相应的Rev蛋白及其抗体.方法:将我国HIV-1 B′/C亚型流行株的rev基因按大肠杆菌优势密码子进行改造后人工合成,在原核系统中与pET30a(+)载体中的His·Tag、Trx·Tag及S·Tag进行融合表达,目的蛋白经Ni2+金属螯合层析柱纯化后用于免疫家兔,制备多克隆抗体.结果与结论:合成基因在原核系统中融合表达得到相对分子质量约18×103的融合蛋白,目的蛋白的表达量约占菌体总蛋白量的36%;用纯化后的融合蛋白免疫家兔,制备了多克隆抗体,Western印迹及间接免疫荧光检测结果显示,获得的多克隆抗体与HIV-1 B′/C亚型的Rev蛋白能产生特异性反应,可用于检测HIV-1 B′/C亚型Rev蛋白的表达.     

Antibody engineering and perspectives in therapy

Techniques of genetic engineering, homologous recombination, and gene transfection make it feasible to produce antigen-binding molecules with widely varying structures. Novel proteins which possess the binding specificity of antibody associated with sequences such as an enzyme or toxin have potential use in immunoassays, in imaging, in immunotherapy. Antibody fusion proteins can also be used as a means to purify proteins or to study the function of surface protooncogenes. This paper reviews the recent data on the obtention and utilisation of the genetically engineered antibody molecules, as well as the approach which consists on the expression in vitro, in Escherichia coli, of a practically unlimited repertoire of Fab fragments and antibody sites.     

Soluble monomeric IgG1 Fc

Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives.     

Evaluation of homo- and hetero-functionally activated glass surfaces for optimized antibody arrays

Antibody arrays hold great promise for biomedical applications, but they are typically manufactured using chemically functionalized surfaces that still require optimization. Here, we describe novel hetero-functionally activated glass surfaces favoring oriented antibody binding for improved performance in protein microarray applications. Antibody arrays manufactured in our facility using the functionalization chemistries described here proved to be reproducible and stable and also showed good signal intensities. As a proof-of-principle of the glass surface functionalization protocols described in this article, we built antibody-based arrays functionalized with different chemistries that enabled the simultaneous detection of 71 human leukocyte membrane differentiation antigens commonly found in peripheral blood mononuclear cells. Such detection is specific and semi-quantitative and can be performed in a single assay under native conditions. In summary, the protocol described here, based on the use of antibody array technology, enabled the concurrent detection of a set of membrane proteins under native conditions in a specific, selective, and semi-quantitative manner and in a single assay.     

抗体在二氧化硅表面固定及活性测定

应用分子自组装技术,通过表面羟基化、氨基硅烷化和对苯二甲醛组装,在二氧化硅表面衍生活泼的醛基基团。利用抗体的氨基和醛基发生还原胺化反应将抗体固定在二氧化硅表面,通过抗原抗体反应定性检测固定抗体的生物活性。结果显示应用这种方法能够有效将抗体固定在二氧化硅表面,并保持抗体生物活性,该固定方法在蛋白质检测、分析和蛋白质芯片中有广泛应用价值。     

Fabrication of an antibody microwell array with self-adhering antibody binding protein

One of the promising methods of preparing antibody arrays is immobilizing antibodies with protein A or protein G, each of which binds specifically to the heavy chain constant (Fc) region of immunoglobulin G (IgG). In this system, antibody immobilization efficiency depends on the number of active Fc binding proteins that need to be immobilized on the surface. Here we have designed and constructed an Fc binding protein with a self-adhering ability that can be immobilized on the hydrophobic surface by simple adsorption. It consists of an Fc binding domain of protein G (G3) and hydrophobic domain of elastin (E72). Direct observation revealed its self-adhering ability on the hydrophobic surface. The enzyme-linked immunosorbent assay (ELISA) showed that it retained antibody binding ability on the surface. The antibody array model was prepared on a hydrophobic microwell glass slide with E72G3, which specifically detect the antigen with a sevenfold greater sensitivity than the G3-treated slide. These results suggest that the E72G3 is useful for simple and effective immobilization of antibodies and can be used to fabricate any immuno devices.     

Engineering a Monomeric Fc Domain Modality by N-Glycosylation for the Half-life Extension of Biotherapeutics

Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic C_H3-C_H3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.     

Development of antibody to measles virus polypeptides during complicated and uncomplicated measles virus infections.

Immune precipitation of 181 sera from 152 patients with natural measles was studied to determine the temporal course and frequency of antibody responses to nucleocapsid, fusion, hemagglutinin, and matrix proteins of measles virus. Large amounts of antibody to nucleocapsid protein developed in all patients by day one of the rash. Antibody to hemagglutinin and fusion proteins developed in all patients over the next 3 weeks, the former to high levels and the latter to low levels. Antibody to matrix protein developed to very low levels and was detectable in only 41% of the patients; this poor response to matrix protein was not correlated with the age of the patient or the acute neurological complications of measles.     

Design of a modular immunotoxin connected by polyionic adapter peptides

Immunotoxins are genetically engineered fusion proteins of an antibody Fv fragment and a toxin from bacteria or plants, which function as anti-cancer therapeutics. Here, we describe a new generation of immunotoxins in which both proteins do not form a single fusion protein but are coupled specifically via cysteine-containing polyionic fusion peptides. The engineered Pseudomonas exotoxin PE38 was N-terminally fused to the peptide E(8)C. In combination with the disulfide-stabilized Fv fragment of the tumor-specific antibody B3, which was extended by the peptide R(8)CP, the fusion peptides ensured a specific and covalent coupling of the Fv fragment and the toxin. The resulting immunotoxin was as active and as specific as an immunotoxin consisting of a fusion protein of the same antibody fragment connected to the toxin.     

抗体固定化方法研究进展

在免疫分析和生物芯片中,抗原-抗体特异性结合被广泛应用,其中抗体的固定化是研发高效诊断和分离工具的关键环节。生物分子工程、材料化学与交联剂化学的进步极大地促进了抗体固定化技术的发展。 抗体可以通过物理吸附、共价偶联和亲和相互作用固定到不同类型的固相表面。 抗体固定化的目标是以一种正确的空间取向将抗体固定到固相表面,在完全保留抗体构象和活性的同时最大化抗原的结合能力,这对固相化抗体的分析性能至关重要。 对固定抗体到固相载体表面的各种最新方法进行了阐述,包括物理吸附法,通过羧基、氨基、巯基、糖基和点击化学的共价结合法以及基于生物亲和作用的固定法,并对固定化抗体的表征方法进行了归纳,最后对抗体固定化方法的发展方向进行了展望。     

新型蛋白质配体-亲和体研究进展

亲和体(affibody)是一种衍生于葡萄球菌A蛋白B结构域的人工蛋白质分子．B结构域含58个氨基酸,形成3个α螺旋结构,分子质量约为6.5 ku．其中第一及第二螺旋中的13个特定位点的氨基酸对其结构无明显影响,这些位点可被随机突变形成理论上可与任何靶分子结合的亲和体文库．筛选该文库可获得能与某一靶分子特异结合的亲和体．亲和体与靶分子的结合特性与抗体相似,但与抗体相比具有一些独特的优势,如：通过体外筛选即可获得,以化学合成方法或原核表达即可大量制备,分子质量小、在生物体内组织穿透性强、血浆清除率高,理化稳定性好,可以通过交联或融合表达与标记分子(如荧光蛋白、生物素等)结合而不影响其与靶分子的结合能力．亲和体可作为抗体的替代品,用于蛋白质识别、分离及纯化、实验诊断、分子显像及靶向治疗．