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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
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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
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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. 相似文献
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胸苷酸合成酶(thymidylate synthase, TS)是催化生物体内胸苷酸合成所必需的酶, 多年来一直作为肿瘤化疗的靶点. 研究表明, TS是一种RNA结合蛋白, 可以与其自身mRNA的2个位点结合, 使mRNA翻译受阻. 本文以mRNA体外展示技术进行了由大容量多肽库(>1013)中筛选胸苷酸合成酶mRNA亲和多肽的研究, 对随机库进行了12轮循环的选择及扩增. 结果表明, 与初始库相比, 经选择循环之后, 碱性氨基酸及芳香族的苯丙氨酸含量明显增加, 它们在TS RNA与蛋白质的相互作用中扮演着重要角色. 按其理化特性对每一随机位点的氨基酸进行分类, 并与初始库比较, 发现位点1, 12, 17和18具有明显的带正电荷的特性, 表明碱性侧链参与了与RNA的结合. 二级结构预测表明, 随着筛选的进行, 与TS mRNA 亲和的多肽显示出明显的螺旋倾向, 而且形成螺旋的区域富含碱性氨基酸. 凝胶迁移及体外翻译实验证实, 选择循环之后的多肽能够与TS mRNA高度亲和, 并能抑制TS mRNA的翻译. 本研究表明mRNA体外展示方法得到的亲和多肽可以用作新的TS RNA的翻译抑制剂, 并有可能成为一类新型的抗肿瘤药物. 相似文献
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噬菌体展示技术的发展及应用 总被引:9,自引:0,他引:9
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… 相似文献
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In vitro peptide and protein selection using mRNA display enables the discovery and directed evolution of new molecules from combinatorial libraries. These selected molecules can serve as tools to control and understand biological processes, enhance our understanding of molecular interactions and potentially treat disease in therapeutic applications. In mRNA display, mRNA molecules are covalently attached to the peptide or protein they encode. These mRNA-protein fusions enable in vitro selection of peptide and protein libraries of >10(13) different sequences. mRNA display has been used to discover novel peptide and protein ligands for RNA, small molecules and proteins, as well as to define cellular interaction partners of proteins and drugs. In addition, several unique applications are possible with mRNA display, including self-assembling protein chips and library construction with unnatural amino acids and chemically modified peptides. 相似文献
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R W Roberts 《Current opinion in chemical biology》1999,3(3):268-273
Both chemists and biochemists have great interest in creating peptides and proteins with desired structure, recognition and catalytic properties. Recently, two techniques have been developed that afford functional selection of proteins entirely in vitro: mRNA-protein fusions, and ribosome display. Improvements in both methods have allowed peptide and protein libraries of unprecedented size (10(11)-10(13) different members) to be generated and screened for function. 相似文献
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Ribosome display: cell-free protein display technology. 总被引:4,自引:0,他引:4
Ribosome display is a cell-free system for the in vitro selection of proteins and peptides from large libraries. It uses the principle of coupling individual nascent proteins (phenotypes) to their corresponding mRNA (genotypes), through the formation of stable protein-ribosome-mRNA (PRM) complexes. This permits the simultaneous isolation of a functional nascent protein, through affinity for a ligand, together with the encoding mRNA, which is then converted and amplified as DNA for further manipulation, including repeated cycles or protein expression. Ribosome display has a number of advantages over cell-based systems such as phage display; in particular, it can display very large libraries without the restriction of bacterial transformation. It is also suitable for generating toxic, proteolytically sensitive and unstable proteins, and allows the incorporation of modified amino acids at defined positions. In combination with polymerase chain reaction (PCR)-based methods, mutations can be introduced efficiently into the selected DNA pool in subsequent cycles, leading to continuous DNA diversification and protein selection (in vitro protein evolution). Both prokaryotic and eukaryotic ribosome display systems have been developed and each has its own distinctive features. In this paper, ribosome display systems and their application in selection and evolution of proteins are reviewed. 相似文献
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Selection of RNA-binding peptides using mRNA-peptide fusions 总被引:4,自引:0,他引:4
We have been working to apply in vitro selection to isolate novel RNA-binding peptides. To do this, we use mRNA-protein fusions, peptides covalently attached to their own mRNA. Here, we report selection protocols developed using the arginine-rich domain of bacteriophage lambda-N protein and its binding target, the boxB RNA. Systematic investigation of possible paths for a selection round has allowed us to design a reliable and efficient protocol to enrich RNA-binding peptides from nonfunctional members of a complex mixture. The protocols we have developed should greatly facilitate the isolation of new molecules using the fusion system. 相似文献
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We describe the synthesis of a novel type of mRNA template and its use in the preparation of mRNA-protein fusions. A light-induced psoralen crosslinking reaction was used to attach a puromycin-containing oligonucleotide to the 3'-end of an mRNA template. The photo-crosslinked template was found to undergo efficient mRNA-protein fusion formation in rabbit reticulocyte lysate. Fusion formation was subsequently tested with templates carrying puromycin linkers of different length and chemical composition. Short linkers with multiple triethyleneglycol phosphate building blocks allowed the most efficient fusion formation under a wide range of salt conditions. The present method simplifies the preparation of mRNA-protein fusions and thus significantly accelerates the in vitro protein evolution procedure which involves repetitive cycles of fusion production and selection. 相似文献
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《Bioorganic & medicinal chemistry letters》2020,30(4):126934
Solid-phase resins functionalized with poly-deoxythymidine (dT) oligos facilitate purification of poly-adenylated molecules from solution through high affinity, high selectivity base-pairing interactions. These resins are commonly used to purify messenger RNA (mRNA) from complex biological mixtures as well as mRNA-protein fusion molecules for mRNA Display selections. Historically, dT-conjugated cellulose was the primary resin for poly-dA purification, but its scarcity has prompted the development of alternative resins, most notably dT-functionalized magnetic beads. In order to develop a cost-effective alternative to commercially available poly-dT resins for large-scale purifications of mRNA-protein fusions, we investigated the purification properties of dT25-conjugated Oligo Affinity Support resin (dT25-OAS) alongside poly-dT14 magnetic beads and dT25-cellulose. dT25-OAS was found to have the highest dA21 oligo binding capacity at 4 pmol/µg, followed by dT14-magnetic beads (1.1 pmol/µg) and dT25-cellulose (0.7 pmol/µg). To determine the resin specificity in the context of a complex biological mixture, we translated mRNA-protein fusions consisting of a radiolabeled Her2 affibody fused to its encoding mRNA. Commercial dT25-cellulose showed the highest mRNA-affibody purification specificity, followed by dT25-OAS and dT14-magnetic beads. Overall, dT25-OAS showed exceptionally high binding capacity and low background binding, making it an attractive alternative for large-scale mRNA purification and mRNA Display library enrichment. 相似文献
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噬菌体展示技术及其在肿瘤研究中的应用 总被引:1,自引:0,他引:1
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。 相似文献