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.     

噬菌体展示系统的研究进展

噬菌体展示系统的研究进展许正平（浙江大学生物科学与技术系,杭州３１００２７）李伯良（中国科学院上海生物化学研究所,上海２０００３１）关键词噬菌体展示系统噬菌体展示是一种将外源肽或蛋白质与特定噬菌体衣壳蛋白融合并展示于噬菌体表面的技术。若展示的是随机肽...     

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.     

噬菌体抗体库技术的研究进展及应用

噬菌体抗体库技术是继噬菌体展示技术发展而来的一项基因抗体工程新技术。它可将含不同物种全部抗体可变区基因的基因库转化成展示在噬菌体表面的蛋白库,不仅使单克隆抗体的生产更方便、快速、高效地在体外进行,还开辟了单克隆抗体人源化的新途径,促进了人类单克隆抗体生产的发展。就近年来噬菌体抗体库技术的基因来源、发展关键及抗体应用的研究作一综述。     

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…     

噬菌体展示技术在疫苗研究中的应用进展

噬菌体是一种以细菌为宿主的具有严格宿主特异性的病毒,近年来由于分子生物学和基因重组技术的长足发展,藉助噬菌体的基本特性创立和发展了噬菌体展示技术,此项技术将外源肽或蛋白与特定噬菌体衣壳蛋白融合并展示于噬菌体表面。利用这项技术制作的疫苗具有安全可靠、稳定性高、免疫效果好等优点,因此,在新型疫苗的研制上具有很大的应用价值。就噬菌体展示技术及其在疫苗研究中的优势、预防性疫苗与治疗性疫苗的研究进展予以综述。     

噬菌体展示定位和噬菌体展示工程

噬菌体展示是指将外源基因克隆到丝状噬菌体ｆｄ染色体ＤＮＡ上,然后以融合白 形式展示了于噬菌体衣壳蛋白表面的技术。本文将概述丝状噬菌体展示技术的最新进展,特别是表位定位的原理及其应用,可包括以下三部分：１．比较几种不同噬菌体展示策略的技术原理;２．展示表位的重组噬菌体在诊断试剂上的开发价值;３．展示表位的重组噬菌体在疫备研究上的应用前景。     

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。     

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。     

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。     

体内筛选技术在靶向治疗中的新进展

用噬菌体展示技术进行体内筛选可以更好地模拟靶抗原的天然环境 ,以筛选到与活体内某些器官或组织有特异结合活性的肽或抗体。近年来利用该技术在动物体内的研究已取得了可喜的进展。综述了体内筛选技术在器官和组织血管靶向载体的筛选、基因治疗及绘制人类血管分子图谱方面的应用 ,并对其今后的研究发展方向进行了阐述。     

Bacteriophage Vehicles for Phage Display: Biology,Mechanism, and Application

The phage display technique is a powerful tool for selection of various biological agents. This technique allows construction of large libraries from the antibody repertoire of different hosts and provides a fast and high-throughput selection method. Specific antibodies can be isolated based on distinctive characteristics from a library consisting of millions of members. These features made phage display technology preferred method for antibody selection and engineering. There are several phage display methods available and each has its unique merits and application. Selection of appropriate display technique requires basic knowledge of available methods and their mechanism. In this review, we describe different phage display techniques, available bacteriophage vehicles, and their mechanism.     

Generation of a murine single chain Fv (scFv) antibody specific for cucumber mosaic virus (CMV) using a phage display library

With the long-term goal of generating CMV-resistant transgenic plants using antibody genes, a single-chain variable fragment (scFv) antibody that binds to the cucumber mosaic virus was isolated from a scFv phage display library by four rounds of affinity selection with CMV-Mf as an antigen. The scFv has the identical binding specificity to CMV as a monoclonal antibody that is generated by the hybridoma fusion technique, and recognized purified preparations of CMV isolates belonging to either subgroup I or II in immunoblotting. The nucleotide sequences of the recombinant antibody showed that a heavy chain variable region (V(H)) gene belonged to the VH3 subgroup and the kappa light chain variable region (V kappa) came from the Vkappa4 subgroup. Our results demonstrate that the scFv phage display library, an alternative approach to the traditional hybridoma fusion technique, has a potential applicability in the study of plant virus and plant pathology.     

Phage display screening of therapeutic peptide for cancer targeting and therapy

Recently, phage display technology has been announced as the recipient of Nobel Prize in Chemistry 2018. Phage display technique allows high affinity target-binding peptides to be selected from a complex mixture pool of billions of displayed peptides on phage in a combinatorial library and could be further enriched through the biopanning process; proving to be a powerful technique in the screening of peptide with high affinity and selectivity. In this review, we will first discuss the modifications in phage display techniques used to isolate various cancer-specific ligands by in situ, in vitro, in vivo, and ex vivo screening methods. We will then discuss prominent examples of solid tumor targeting-peptides; namely peptide targeting tumor vasculature, tumor microenvironment (TME) and overexpressed receptors on cancer cells identified through phage display screening. We will also discuss the current challenges and future outlook for targeting peptidebased therapeutics in the clinics.     

Genetic selection of phage engineered for receptor-mediated gene transfer to mammalian cells.

Although phage display is a powerful way of selecting ligands against purified target proteins, it is less effective for selecting functional ligands for complex targets like living cells. Accordingly, phage display has had limited utility in the development of targeting agents for gene therapy vectors. By adapting a filamentous bacteriophage for gene delivery to mammalian cells, however, we show here that it is possible to screen phage libraries for functional ligands capable of delivering DNA to cells. For example, when targeted with epidermal growth factor (EGF), M13 bacteriophage were capable of delivering a green fluorescent protein (GFP) gene to EGF receptor bearing cells in a ligand-, time-, and phage concentration-dependent manner. The EGF-targeted phage transduced COS-1 cells in a highly specific manner as demonstrated by competition with excess free EGF or alternatively with anti-EGF receptor antibodies. We further demonstrate that EGF-phage can be selected, by their ability to transduce EGF receptor bearing cells from libraries of peptide display phage. When phage were incubated with COS-1 cells, EGF ligand-encoding sequences were recovered by PCR from FACsorted, GFP-positive cells and the EGF-displaying phage were enriched 1 million-fold by four rounds of selection. These data suggest the feasibility of applying molecular evolution to phage gene delivery to select novel cell-specific DNA-targeting ligands. The same approach could be used to select genetically altered phage that are specifically designed and evolved as gene therapy vectors.     

Phage display of functional, full-length human and viral membrane proteins

Phage display of protein and peptide libraries offers a powerful technology for the selection and isolation of ligands and receptors. To date, the technique has been considered limited to soluble, non-membrane proteins. We report two examples of phage display of full-length, folded and functional membrane proteins. Consistent display required the recently reported KO7(+) helper phage. The two proteins, full-length caveolin-1 and HIV gp41, display well on the surface of the phage, and maintain their binding activities as shown by in vitro assays.     

Bacteriophage lambda display systems: developments and applications

Bacteriophage (phage) Lambda (λ) has played a key historic role in driving our understanding of molecular genetics. The lytic nature of λ and the conformation of its major capsid protein gpD in capsid assembly offer several advantages as a phage display candidate. The unique formation of the λ capsid and the potential to exploit gpD in the design of controlled phage decoration will benefit future applications of λ display where steric hindrance and avidity are of great concern. Here, we review the recent developments in phage display technologies with phage λ and explore some key applications of this technology including vaccine delivery, gene transfer, bio-detection, and bio-control.     

Construction and exploitation in model experiments of functional selection of a landscape library expressed from a phagemid

Phage display has evolved during the past 15 years as a powerful technique to select, from libraries of peptides or proteins, binders for various targets or to evolve new functions in proteins. In recent years, the knowledge acquired in phage display technology was exploited to engineer phages as vehicles for receptor-mediated gene delivery. The first vectors generated provided the proof of the concept that development of gene delivery vehicles based on phages was feasible. Results obtained showed that the level of receptor ligand display was an essential factor that determines the efficiency of transduction and suggested that phagemids might be more appropriate than phages for gene delivery. However, due to the limitations of the existing display systems, vectors constructed up to now allowed only relatively low levels of ligand display. The transduction efficiency of these vectors was relatively poor. Here, we describe the construction and optimization of a new phagemid display system that was designed to allow the functional selection of peptides that promote gene delivery from phagemids in a high display format. Peptides are displayed on every copy of the major coat protein pVIII and are expressed from the phagemid itself. The phagemid is rescued as particles by a modified R408 helper phage, deficient in pVIII production. Besides an expression cassette for pVIII, the phagemid also contains the SV40 origin of replication, the GFP gene and the neomycin resistance marker. As a model we constructed a library of octapeptides and showed that the library is amenable to selection on cos-7 cells. Several selection approaches were investigated and a preliminary analysis of the peptides selected was carried out.     

噬菌体展示技术的理论基础及其在感染性疾病防治中的应用

噬菌体展示技术是一种高通量研究基因功能、蛋白质表达及其相互作用的有效方法,具有与生物芯片一样的大量快速检测、发现并鉴定功能基因及蛋白质-蛋白质相互作用的优点。我们着重介绍噬菌体展示技术的种类、原理,及其在感染性疾病防治中的应用。     

Effect of DNA copy number on genetic stability of phage-displayed peptides

A small model peptide, the FLAG epitope, was cloned into two filamentous phage display vectors, f88-4 and fd88-4, creating phages f88-FLAG and fd88-FLAG, respectively. Both vectors have a gene VIII display cassette (in addition to their normal phage gene VIII) and display the cloned peptide on a few percent of the virion's 3000-4000 pVIII (major coat protein) subunits. Vector f88-4 has a replication defect and attains low DNA copy number in infected cells, while vector fd88-4 has no replication defect and attains the normal, high DNA copy number characteristic of wild-type filamentous phage. Almost no loss of displayed peptide was observed during six rounds of propagation of low copy number f88-FLAG phage. In contrast, when high copy number fd88-FLAG phage was similarly propagated, variant clones that did not display the FLAG epitope accumulated gradually. The loss of displayed peptide from the high copy number vector is undoubtedly slow enough to be overcome by even weak affinity selection, and high copy number vectors have important advantages that make their use worth considering, at least when the displayed peptides are small.