噬菌体展示技术的发展及应用

噬菌体展示技术是一种用于筛选和改造功能性多肽的生物技术 ,编码多肽的ＤＮＡ片段与噬菌体表面蛋白的编码基因融合后 ,以融合蛋白的形式在噬菌体的表面表达出多肽序列。这是一种表型与基因型的统一。噬菌体展示技术最初是以Ｍ 13噬菌体为载体的 ,其宿主菌为大肠杆菌。以大肠杆菌为宿主的展示系统还有其他 ,如λ噬菌体和Ｔ4噬菌体等展示系统。还有利用真核细胞的病毒以及酵母菌作为展示系统的。这些展示系统各有各的优势 ,但最常用的仍是Ｍ 13噬菌体表达系统。最初的噬菌体展示系统是将外源肽或蛋白质与噬菌体外壳蛋白ＰⅢ或ＰⅧ的Ｎ末端融…     

T4噬菌体表面展示技术的研究进展

噬菌体表面展示技术(phage display)是由Smith于1985年首先建立起来的一种新的生物技术[1],它能将表达的外源多肽或蛋白以融合蛋白的形式展示在噬菌体的表面,保持相对独立的空间构象和原有的生物活性[2].常用的噬菌体表面展示系统主要有丝状噬菌体、λ噬菌体及T4噬菌体展示系统等.虽然它们都具有噬菌体展示系统的优点,但对于丝状噬菌体来说,它不能展示那些难以分泌的肽和蛋白质,而且它的N端可融合外源多肽的容量有限,较大蛋白的融合会造成空间障碍,影响噬菌体的装配,使其失去感染力.而对于λ噬菌体,大分子蛋白的融合会抑制噬菌体的组装,使其生长受到影响,因此这两种噬菌体更适用于构建短肽库和cDNA表达文库[3],而不适于构建重组疫苗和表达分子量大具有完整结构域的蛋白质[4,5].     

噬菌体展示技术研究进展

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。     

噬菌体展示技术的原理和方法

噬菌体展示(Phage display)最早是由George开始于 1 985年1.最先构建的噬菌体多肽或抗体展示文库则始于 1990年2,3.由此,噬菌体展示技术进入了一个飞速发展的时期。噬菌体展示的基本概念是将外源蛋白质或多肽的基因表达产物与噬菌体衣壳蛋白融合,并在其表面展示,同时将其遗传密码信息整合到个体噬菌体的基因组中。这个技术的最大优点是直接将可现的表达型与其基因型联系在一起,再利用其配体的特异性亲和力,将所感兴趣的蛋白质或多肽挑选出来。       

噬菌体展示技术及其在肿瘤研究中的应用

噬菌体表面展示技术是一项特异性多肽或蛋白的筛选技术,它将随机序列的多肽或蛋白片段与噬菌体衣壳蛋白融合表达而呈现于病毒表面,被展示的多肽能保持相对独立的空间结构,使其能够与配体作用而达到模仿性筛选特异性分子表位,从而提供了高通量高效率的筛选系统。近年来噬菌体展示技术已广泛应用于肿瘤抗原抗体库的建立、单克隆抗体制备、多肽筛选、疫苗研制、肿瘤相关抗原筛选和抗原表位研究、药物设计、癌症检测和诊断、基因治疗及细胞信号转导研究等。就近年来噬菌体展示技术在肿瘤相关研究中的运用作以综述。     

噬菌体展示技术及其在寄生虫研究中的应用

噬菌体展示技术是将外源蛋白或多肽的编码基因或DNA序列插入到噬菌体外壳蛋白结构基因的适当位置,使外源基因随外壳蛋白的表达而表达,并随噬菌体的重新组装而展示到噬菌体表面的生物技术.在研究蛋白质识别或蛋白质与核酸相互作用的生物学过程、蛋白质定向改造、研制新型多肽药物、疫苗和抗体等多领域具有重要作用.就噬菌体展示技术基本原理及特点,以及噬菌体展示技术在寄生虫研究中的应用做一简要综述.     

噬菌体展示文库筛选技术的研究进展

噬菌体展示技术是将编码外源蛋白或多肽的基因片段定向插入到噬菌体的外壳蛋白基因区,使外源蛋白或多肽通过与噬菌体外壳蛋白融合而表达并展示于噬菌体表面,进而筛选表达特异蛋白或多肽的噬菌体,已发展成为生物学后基因组时代一个强有力的实验技术.噬菌体展示文库的筛选是其关键环节.为了提高筛选效率,许多研究者对传统的筛选技术进行了改进,如选择性感染噬菌体、迟延感染性噬菌体、以DNA为基础的筛选方法、亲合力捕获和反复筛选和封闭筛选法等,用于筛选的靶标也越来越具有多样性,使得这一技术有了更加广阔的发展前景.     

噬菌体表面展示技术

噬菌体展示技术(PhageDisplayTechniques,PDT)是一种用于筛选和改造功能性多肽的生物技术。自问世以来,它已被广泛应用于生命科学的许多领域。对噬菌体展示技术的基本原理、噬菌体表面展示系统研究以及噬菌体展示技术的应用、展望等进行了探讨,由此可以看出:噬菌体展示技术的进一步发展,必将为生命科学及相关学科的发展带来深远的影响。     

辅助噬菌体在噬菌体展示中的应用及研究进展

噬菌体展示技术是一种将外源肽或蛋白质与特定噬菌体衣壳蛋白相融合,展示于噬菌体表面来构建蛋白质或多肽文库,并从中筛选目的蛋白、多肽或抗体的基因工程高新技术。噬菌粒/辅助噬菌体系统是最常用的噬菌体展示系统,此系统中辅助噬菌体对噬菌粒的复制和组装发挥着至关重要的作用。本文结合当今该领域的最新研究动态,概述了噬菌粒和辅助噬菌体双基因组系统,着重介绍了不同辅助噬菌体的特点及其突变机制,并对其应用前景进行了展望,以期为该技术的进一步完善提供一定的借鉴作用。     

噬菌体展示技术发展

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。     

Bacterial display in combinatorial protein engineering

Technologies for display of recombinant protein libraries are today essential tools in many research-intensive fields, such as in the drug discovery processes of biopharmaceutical development. Phage display is still the most widely used method, but alternative systems are available and are becoming increasingly popular. The most rapidly expanding of the alternative systems are the cell display-based technologies, offering innovative strategies for selection and characterization of affinity proteins. Most investigations have focused on eukaryotic yeast for display of protein libraries, but similar systems are also being developed using prokaryotic hosts. This review summarizes the field of bacterial surface display with a strong emphasis on library applications for generation of new affinity proteins. The main focus will be on the most recent progress of the work on primarily Escherichia coli, but also on studies using a recently developed system for display on Gram-positive Staphylococcus carnosus. In addition, general strategies for combinatorial protein engineering using cell display are discussed along with the latest developments of new methodologies with comparisons to mainly phage display technology.     

Display of proteins on bacteria

Display of heterologous proteins on the surface of microorganisms, enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and vaccinology. Gram-negative, Gram-positive bacteria, viruses and phages are all being investigated in such applications. This review will focus on the bacterial display systems and applications. Live bacterial vaccine delivery vehicles are being developed through the surface display of foreign antigens on the bacterial surfaces. In this field, 'second generation' vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals, through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. Certain bacteria have also been employed for display of various poly-peptide libraries for use as devices in in vitro selection applications. Through various selection principles, individual clones with desired properties can be selected from such libraries. This article explains the basic principles of the different bacterial display systems, and discusses current uses and possible future trends of these emerging technologies.     

Stark sexual display divergence among jumping spider populations in the face of gene flow

Gene flow can inhibit evolutionary divergence by eroding genetic differences between populations. A current aim in speciation research is to identify conditions in which selection overcomes this process. We focused on a state of limited differentiation, asking whether selection enables divergence with gene flow in a set of Habronattus americanus jumping spider populations that exhibit three distinct male sexual display morphs. We found that each population is at high frequency or fixed for a single morph. These strong phenotypic differences contrast with low divergence at 210 AFLP markers, suggesting selection has driven or maintains morph divergence. Coinciding patterns of isolation by distance and ‘isolation by phenotype’ (i.e. increased genetic divergence among phenotypically contrasting populations) across the study area support several alternative demographic hypotheses for display divergence, each of which entails gene flow. Display‐associated structure appears broadly distributed across the genome and the markers producing this pattern do not stand out from background levels of differentiation. Overall, the results suggest selection can promote stark sexual display divergence in the face of gene flow among closely related populations.     

Ribosome display: next-generation display technologies for production of antibodies in vitro

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.     

外源表位在Salmonella菌毛上的表达

过去的20年中,在细菌表面展示外源多肽的表达系统的研究取得了重要进展。而其中相当一部分是以细菌菌毛作为表达载体用于表达外源多肽或蛋白。本文将详述一种特殊的利用基因置换构建的沙门菌菌毛外源多肽展示系统,同时介绍一些其他的菌毛展示系统并探讨他们的优劣性。     

Biotechnology techniques for the development of new tumor specific peptides

Peptides, proteins and antibodies are promising candidates as carriers for radionuclides in endoradiotherapy. This novel class of pharmaceuticals offers a great potential for the targeted therapy of cancer. The fact that some receptors are overexpressed in several tumor types and can be targeted by small peptides, proteins or antibodies conjugated to radionuclides has been used in the past for the development of peptide endoradiotherapeutic agents such as ⁹⁰Y-DOTATOC or radioimmunotherapy of lymphomas with Zevalin. These procedures have been shown to be powerful options for the treatment of cancer patients.Design of new peptide libraries and scaffolds combined with biopanning techniques like phage and ribosome display may lead to the discovery of new specific ligands for target structures overexpressed in malignant tumors. Display methods are high throughput systems which select for high affinity binders. These methods allow the screening of a vast amount of potential binding motifs which may be exposed to either cells overexpressing the target structures or in a cell-free system to the protein itself. Labelling these binders with radionuclides creates new potential tracers for application in diagnosis and endoradiotherapy. This review highlights the advantages and problems of phage and ribosome display for the identification and evaluation of new tumor specific peptides.     

Emerging trends in the synthesis and improvement of hapten-specific recombinant antibodies

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.     

酵母细胞表面展示系统的研究进展及其应用

酵母表达体系不但有原核表达体系的特点,同时具有真核细胞翻译后蛋白加工修饰的过程,因此,以酵母为基础的表面展示体系在诸多展示系统中有独特的优势。将酶,抗原,抗体和六聚组氨酸等不同蛋白和多肽展示在酵母细胞表面,可实现各种各样的用途。本文主要概述了酵母细胞展示的理论基础、展示体系类型及应用研究的进展。