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1.
蛋白质体外表达与进化技术包括核糖体展示技术和mRNA展示技术。与蛋白质体内表达系统相比,体外表达技术可产生较大容量的蛋白质文库(约10^12~10^13左右),同时在回收编码蛋白质的信息时对文库进行了进化,增加了蛋白质文库的多样性,促进了抗体或配体类蛋白质的亲和成熟能力。该文介绍了蛋白质体外表达技术在新蛋白质(如抗体或配体等)表达筛选中的应用。  相似文献   

2.
核糖体展示是一种无细胞系统,可以从文库中筛选蛋白质和多肽。翻译的蛋白质及其mRNA同时结合在核糖体上形成mRNA-核糖体-蛋白质三聚体,通过配体亲和分离得到功能性蛋白及其编码的mRNA,转换成对应的DNA后进行相关蛋白的表达,可用于抗体及蛋白质文库选择、蛋白质体外改造等,而且其可以展示较大的文库而不受细菌转化的限制,可对毒蛋白、蛋白酶敏感和不稳定的蛋白质进行筛选,也可在特定位点进行氨基酸修饰。就核糖体展示技术的研究进展及其在蛋白质进化和筛选方面的应用进行综述。  相似文献   

3.
噬菌体表面展示技术是一种将外源蛋白或抗体可变区与噬菌体表面特定蛋白质融合并展示于其表面,构建蛋白质或抗体库,并从中筛选特异蛋白质或抗体的基因工程技术。介绍这一技术的原理、相关展示系统以及在蛋白质相互作用的研究,抗体及疫苗的制备、多肽药物的研制等方面的应用潜力和独特的优点。  相似文献   

4.
核糖体展示及体外分子选择与进化   总被引:4,自引:0,他引:4  
核糖体展示是20世纪90年代中期发展起来的一种简便而有效的体外分子选择与进化技术。它也是第一种完全在体外进行蛋白质或多肽分子选择与进化的方法。本主要概述了体外核糖体展示技术的建立基础、基本原理和技术特点等,并跟踪了目前该领域的最新研究进展和发展前景。  相似文献   

5.
噬菌体展示技术发展   总被引:1,自引:0,他引:1  
噬菌体表面展示技术是一种将外源蛋白或抗体可变区与噬菌体表面特定蛋白质融合并展示于其表面,构建蛋白质或抗体库,并从中筛选特异蛋白质或抗体的基因工程技术。随着该项技术的不断完善和发展,噬菌体展示技术已被广泛应用于生命科学研究的不同领域,并显示了良好的应用前景。  相似文献   

6.
噬菌体展示技术发展   总被引:1,自引:0,他引:1  
罗扬拓  朱承睿  武元  李骢 《生物磁学》2011,(12):2389-2390,2349
噬菌体表面展示技术是一种将外源蛋白或抗体可变区与噬菌体表面特定蛋白质融合并展示于其表面,构建蛋白质或抗体库,并从中筛选特异蛋白质或抗体的基因工程技术。随着该项技术的不断完善和发展,噬菌体展示技术已被广泛应用于生命科学研究的不同领域,并显示了良好的应用前景。  相似文献   

7.
抗体库的起源、发展及应用前景   总被引:1,自引:1,他引:0  
抗体是高等动物特异性免疫应答反应所产生的免疫球蛋白,负责特异抗原的识别和清除。抗体不仅是机体抵抗病原体入侵的强大武器,也是基础科学研究中用于特异性分子识别的专用工具。抗体分子的多样性导致了抗体库概念的产生,让我们认识到每个高等生物个体都是一个天然的抗体库。在后基因组时代,为了适应各种“组学”研究,特别是为了蛋白质组学研究的高通量技术需求,在噬菌体展示技术平台的基础上,构建了各种基因工程抗体库和抗体替代物库。但现在越来越多的其他展示技术如核糖体展示、mRNA展示等体外展示技术也被用于抗体库的研究,而且表现出了相比于噬菌体展示更多的优势。以下根据目前最新发表的有关综述文章和研究论文,对抗体库的起源、发展及应用前景给予粗略的描述,为读者提供最新的参考文献,通过分析目前存在的问题,论述了抗体库技术的应用前景和发展趋势。  相似文献   

8.
运用 mRNA 体外展示技术筛选胸苷酸合成酶 RNA 亲和肽   总被引:3,自引:0,他引:3  
以体外选择方法筛选不同功能的核酸、肽和蛋白质是近年的研究热点, mRNA 体外展示是一种新兴的高效多肽选择技术,其基本原理是通过含嘌呤霉素寡核苷酸的 Linker 使 mRNA 与它编码的肽或蛋白质共价结合,形成 mRNA- 蛋白质融合体,这一方法已用于多种功能肽的鉴定 . 以 mRNA 体外展示技术进行了由大容量多肽库中 (>1013) 筛选胸苷酸合成酶 (thymidylate synthase , TS) RNA 亲和肽的研究,通过精密的实验设计,建立了一套完整有效的筛选方法,并对实验条件进行了优化 . 已进行了 8 轮筛选,结果表明,以 mRNA 体外展示技术获得的多肽分子,可以与 TS mRNA 亲和 . 将测序结果与初始肽库进行比较,发现亲和肽中碱性氨基酸及芳香族氨基酸含量明显增加,说明其在与 RNA 结合中具有重要作用 . mRNA 展示技术作为一种大容量文库的体外筛选方法,将广泛应用于与固定化靶物质具高度亲和性及特异性的多肽和蛋白质的筛选 .  相似文献   

9.
纳米抗体(nanobody, Nb)是在骆驼科血清中发现的一种新型抗体,具有体积小、特异性强、稳定性高、易于表达和能识别隐藏的抗原表位等优势,在各个领域具有广泛的应用价值。本文介绍了纳米抗体筛选与优化过程,包括纳米抗体文库构建、体外展示和亲和力成熟3个重要技术阶段的分类与特点。其中,简要描述了天然、免疫及半合成/合成文库的制备方法与重要参数,并系统介绍了应用噬菌体、酵母、细菌、核糖体/mRNA和真核细胞等表面展示系统,以及酵母双杂交、高通量测序和质谱鉴定方法,共8种不同体外展示技术进行快速筛选的方法及其优缺点,汇总用于提升纳米抗体功能可靠性的体外及计算机辅助亲和力成熟技术平台,为综合运用各种技术手段快速获得稳定、可靠、特异的纳米抗体类药物或诊断制剂提供了参考。  相似文献   

10.
文库筛选与分子进化的核糖体展示新方法   总被引:3,自引:0,他引:3  
利用适当的文库筛选技术快速、简便地从DNA文库、随机肽库、抗体库或其它蛋白文库中筛选生物活性物质是目前分子生物学研究的一个热点.核糖体展示是一种完全离体进行的功能蛋白筛选和进化鉴定的新技术,避免了传统的活体筛选技术的缺陷,使得文库容量增大、分子多样性加强.本文系统地评述了核糖体展示技术在制备ScFv单链抗体方面的应用,包括ScFv单链抗体模板的构建、体外转录与体外翻译、亲和筛选及筛选效率的测定以及分子多样性和体外进化研究,讨论了核糖体展示技术目前的发展动态、存在问题及发展趋势.  相似文献   

11.
Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.  相似文献   

12.
Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.  相似文献   

13.
In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.  相似文献   

14.
《MABS-AUSTIN》2013,5(1):236-245
In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.  相似文献   

15.
In vitro display technologies, best exemplified by phage and yeast display, were first described for the selection of antibodies some 20 years ago. Since then, many antibodies have been selected and improved upon using these methods. Although it is not widely recognized, many of the antibodies derived using in vitro display methods have properties that would be extremely difficult, if not impossible, to obtain by immunizing animals. The first antibodies derived using in vitro display methods are now in the clinic, with many more waiting in the wings. Unlike immunization, in vitro display permits the use of defined selection conditions and provides immediate availability of the sequence encoding the antibody. The amenability of in vitro display to high-throughput applications broadens the prospects for their wider use in basic and applied research.  相似文献   

16.
Biotechnological applications of phage and cell display   总被引:20,自引:0,他引:20  
In recent years, the use of surface-display vectors for displaying polypeptides on the surface of bacteriophage and bacteria, combined with in vitro selection technologies, has transformed the way in which we generate and manipulate ligands, such as enzymes, antibodies and peptides. Phage display is based on expressing recombinant proteins or peptides fused to a phage coat protein. Bacterial display is based on expressing recombinant proteins fused to sorting signals that direct their incorporation on the cell surface. In both systems, the genetic information encoding for the displayed molecule is physically linked to its product via the displaying particle. Using these two complementary technologies, we are now able to design repertoires of ligands from scratch and use the power of affinity selection to select those ligands having the desired (biological) properties from a large excess of irrelevant ones. With phage display, tailor-made proteins (fused peptides, antibodies, enzymes, DNA-binding proteins) may be synthesized and selected to acquire the desired catalytic properties or affinity of binding and specificity for in vitro and in vivo diagnosis, for immunotherapy of human disease or for biocatalysis. Bacterial surface display has found a range of applications in the expression of various antigenic determinants, heterologous enzymes, single-chain antibodies, and combinatorial peptide libraries. This review explains the basis of phage and bacterial surface display and discusses the contributions made by these two leading technologies to biotechnological applications. This review focuses mainly on three areas where phage and cell display have had the greatest impact, namely, antibody engineering, enzyme technology and vaccine development.  相似文献   

17.
Combinatory antibody library display technologies have been invented and successfully implemented for the selection and engineering of therapeutic antibodies. Precise targeting of important epitopes on the protein of interest is essential for such isolated antibodies to serve as effective modulators of molecular interactions. We developed a strategy to efficiently isolate antibodies against a specific epitope on a target protein from a yeast display antibody library using dengue virus envelope protein domain III as a model target. A domain III mutant protein with a key mutation inside a cross-reactive neutralizing epitope was designed, expressed, and used in the competitive panning of a yeast display naïve antibody library. All the yeast display antibodies that bound to the wild type domain III but not to the mutant were selectively sorted and characterized. Two unique clones were identified and showed cross-reactive binding to envelope protein domain IIIs from different serotypes. Epitope mapping of one of the antibodies confirmed that its epitope overlapped with the intended neutralizing epitope. This novel approach has implications for many areas of research where the isolation of epitope-specific antibodies is desired, such as selecting antibodies against conserved epitope(s) of viral envelope proteins from a library containing high titer, high affinity non-neutralizing antibodies, and targeting unique epitopes on cancer-related proteins.  相似文献   

18.
In vitro display technologies, such as mRNA display and DNA display are powerful tools to screen peptides and proteins with desired functions from combinatorial libraries in the fields of directed protein evolution and proteomics. When screening combinatorial libraries of polypeptides (phenotype), each of which is displayed on its gene (genotype), the problem remains, how best to recover the genotype moiety whose phenotype moiety has bound to the desired target. Here, we describe the use of a photocleavable 2-nitrobenzyl linker between genotype (DNA or mRNA) and phenotype (protein) in our DNA and mRNA display systems. This technique allows rapid and efficient recovery of selected nucleic acids by simple UV irradiation at 4 degrees C for 15 min. Further, we confirmed that the photocleavable DNA display and mRNA display systems are useful for in vitro selection of epitope peptides, recombinant antibodies, and drug-receptor interactions. Thus, these improved methods should be useful in therapeutics and diagnostics, e.g., for screening high-affinity binders, such as enzyme inhibitors and recombinant antibodies from random peptide and antibody libraries, as well as for screening drug-protein interactions from cDNA libraries.  相似文献   

19.
A key requirement for successful immunotherapeutic and immunodiagnostic applications is the availability of antibodies with high affinity and specificity. In the past, polyclonal antibodies from hyperimmunized animals or monoclonal antibodies from hybridoma cell lines were used extensively and profitably in medicine and immunotechnology. Antibody-based diagnostics, such as immunoassays, are also widely accepted because of their high sensitivity and ease of use as compared to conventional chromatographic techniques. While immunoassays have been used to monitor organic chemical contaminants such as pesticides, food preservatives, antibiotics in agricultural and food industries, hapten-specific antibodies with the desired affinity and specificity are generally difficult to obtain. With the advent of recombinant DNA technology, antibody genes can be amplified and selected through phage display, cell surface display, or cell-free display systems. A particularly useful feature common to all these display systems is the linking of the phenotype and genotype of antibodies during selection. This allows easy co-selection of the desired antibodies and their encoding genes based on the binding characteristics of the displayed antibodies. The selected antibody DNA can be further manipulated for high-level expression, post-translation modification, and/or affinity and specificity improvement to suit their particular applications. Several hapten-specific antibodies, which were successfully selected and engineered to high specificity and affinity using display technologies, have been found to be amenable to conventional immunoassay development. In this review, we will examine different formats of immunoassays designed for hapten identification and various display technologies available for antibody selection and improvement.  相似文献   

20.
The ability of monoclonal antibodies (mAbs) to target specific antigens with high precision has led to an increasing demand to generate them for therapeutic use in many disease areas. Historically, the discovery of therapeutic mAbs has relied upon the immunization of mammals and various in vitro display technologies. While the routine immunization of rodents yields clones that are stable in serum and have been selected against vast arrays of endogenous, non-target self-antigens, it is often difficult to obtain species cross-reactive mAbs owing to the generally high sequence similarity shared across human antigens and their mammalian orthologs. In vitro display technologies bypass this limitation, but lack an in vivo screening mechanism, and thus may potentially generate mAbs with undesirable binding specificity and stability issues. Chicken immunization is emerging as an attractive mAb discovery method because it combines the benefits of both in vivo and in vitro display methods. Since chickens are phylogenetically separated from mammals, their proteins share less sequence homology with those of humans, so human proteins are often immunogenic and can readily elicit rodent cross-reactive clones, which are necessary for in vivo proof of mechanism studies. Here, we compare the binding characteristics of mAbs isolated from chicken immunization, mouse immunization, and phage display of human antibody libraries. Our results show that chicken-derived mAbs not only recapitulate the kinetic diversity of mAbs sourced from other methods, but appear to offer an expanded repertoire of epitopes. Further, chicken-derived mAbs can bind their native serum antigen with very high affinity, highlighting their therapeutic potential.  相似文献   

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