噬菌体展示文库及其应用

近十几年来,噬菌体展示技术得到了迅速的发展。通过展示随机肽库可用来筛选与特殊靶分子相结合的配基;模拟非蛋白的配基;也可用作确定抗体表位的工具。展示蛋白;或其功能结构的文库为我们提供了分析结构与功能关系的体系,并能产生具有改变结合位点或新的催化活性的蛋白。展示短的抗原决定簇的融合噬菌体为开发新的疫苗提供了基础,而表达抗体片段的文库则提供了一种产生单克隆抗体的方法。     

构建噬菌体展示的β转角多肽文库

可以形成I型 β转角构象的多肽CX2 GPX4 C融合表达于丝状噬菌体fd的次要衣壳蛋白 g3p的N端 ,从而展示在噬菌体的表面。构建的多肽文库容量达到 1.0 4× 10 8个。随机挑取了 19个克隆 ,序列分析表明 ,核苷酸和氨基酸的分布与预期的基本一致。19个多肽的疏水性和等电点的综合指标分布广泛。以单克隆抗体 12CA5为靶分子 ,经过 3轮筛选 ,出现明显富集。噬菌体酶联免疫吸附法 (ELISA)以及竞争性ELISA的结果表明 ,从第 3轮洗脱液中随机挑选的 15个噬菌体克隆都能结合于抗体的抗原结合位点。破坏多肽的构象 ,这种结合将丧失     

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。       

红莲型水稻细胞质雄性不育花药蛋白质组学初步分析

采用固相pH梯度－SDS PAGE 双向电泳对红莲型细胞质雄性不育水稻(YTA)的不育系和保持系(YTB)单核期花粉总蛋白质进行了分离,通过银染显色,获得了分辨率和重复性较好的双向电泳图谱。Image Master 2D V5.0 软件可识别约1800个蛋白质点,其中差异表达的蛋白质点数为85。将其中16个差异点采用基质辅助激光解析电离飞行时间质谱（matrix assisted laser desorption/ionizaton time of flight mass spectrometry, MALDI-TOF-MS）进行了肽质指纹图分析,通过采用Mascot 软件对MSDB数据库查询,其中9个蛋白质点得到了鉴定。YTA相对于YTB有部分参与碳代谢和淀粉合成的酶缺失或表达量降低,这些蛋白质分别是ADP-葡萄糖磷酸转移酶（AGPase）,UDP-葡萄糖醛酸脱羧酶,乙酰辅酶A合成酶和二氢硫辛酸脱氢酶等。其中AGPase是参与淀粉合成的蛋白,与花粉发育密切相关。乙酰辅酶A合成酶和二氢硫辛酸脱氢酶是细胞内合成乙酰辅酶A的重要酶,而乙酰辅酶A是进入TCA循环的重要底物,乙酰辅酶A的缺乏可以导致TCA循环不能顺利进行,从而不能提供小孢子发育所需要的大量能量。YTA相对于YTB部分参与碳水化合物代谢的重要酶缺失或表达量降低,有可能导致因线粒体提供的能量不足,淀粉合成受阻,因而花粉不能正常发育。      

BLyS改组噬菌体文库的构建及初步筛选

从质粒pT7474-BLyS及人胎脑cDNA文库中分别扩增出BLyS和APRIL基因,用DNase I消化后,回收小于50 bp的片断,用于DNA改组。PCR产物与噬菌体载体pfUSE5连接后,电转E.coliER2738获得改组文库。构建的改组文库库容量为8.9×105。对文库进行初步的筛选,获得了一个受体结合活性降低的突变克隆。成功构建了BLyS改组噬菌体文库,为蛋白结构与功能之间关系的深入研究打下了基础。     

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…     

分子文库展示技术

分子文库展示技术是一系列广泛应用于多肽、蛋白质及药物筛选和研究蛋白质间相互作用的有效的生物学技术。它将组合成的具有一定长度的随机序列寡核苷酸片段(或cDNA)克隆到特定表达载体中,使其表达产物(多肽片段或蛋白质结构域)以融合蛋白的形式展示在活的噬菌体或细胞表面。根据其蛋白质表达是否依赖于宿主表达系统,分为体内表达展示系统和无细胞展示系统(体外表达展示系统)。就其展示的部位不同又可分为噬菌体展示技术、细胞表面展示技术、核糖体展示技术、mRNA展示技术等。现对各种展示技术的基本原理及相关应用做简要综述。     

人尿道上皮细胞T7噬菌体展示cDNA文库的构建与鉴定

[目的]构建人尿道上皮细胞(SV-HUC-1)T7噬菌体展示c DNA文库,为研究人尿道上皮细胞与生殖道感染病原体的相互作用奠定基础。[方法]用Trizol试剂提取SV-HUC-1细胞总RNA,分离纯化出mRNA,经反转录合成得到其双链c DNA,在双链c DNA末端加上定向的EcoRⅠ/HindⅢ黏性末端,然后收集并纯化200bp以上的双链c DNA片段,连接于T7噬菌体载体,经体外包装后转入BLT5403宿主菌,T7噬菌体展示c DNA文库构建成功。[结果]将文库扩增后,用噬斑试验检测其库容,结果为1.2×106pfu/cm3。用PCR鉴定随机挑取的噬菌斑,计算其重组率达93.75%,且插入片段都大于200bp。[结论]成功构建了SV-HUC-1细胞T7噬菌体展示c DNA文库,为下一步研究泌尿生殖道感染病原体与人尿道上皮细胞的相互作用奠定了前期实验基础。     

利用氨甲蝶呤－琼脂糖凝胶进行噬菌体展示人肝脏cDNA文库筛选的方法研究

目的：利用氨甲蝶呤（MTX）偶联琼脂糖凝胶吸附法从人肝脏细胞cDNA噬菌体展示文库中筛选与MTX相互作用的蛋白。方法：以偶联于琼脂糖凝胶表面的MTX为配基,通过"结合－洗脱－扩增"过程筛选与MTX相互作用的噬菌体。利用PCR对筛选结果进行监测,对筛选得到的噬菌体PCR产物进行序列测定和基因同源性分析。结果：通过五轮亲和筛选富集到特异噬菌体克隆,再通过PCR获得cDNA插入片段。通过BLAST程序搜索GenBank,证明筛选到的片段与人PI-3K相关蛋白激酶 SMG-1异构体1 蛋白同源性达100％。结论：利用偶联MTX的琼脂糖凝胶作为筛选基质,从T7噬菌体展示cDNA文库中富集特异噬菌体是一种方便、高效的MTX相互作用靶蛋白筛选方法。本方法可为探讨小分子药物的分子作用机制提供借鉴和参考.     

A DNA-binding peptide from a phage display library

A DNA-binding peptide was selected from a random peptide phage display library. For competitive elution using the DNA methyltransferase M.TaqI in the selection step, a biotin-labeled duplex oligodeoxyribonucleotide containing the 5'-TCGA-3' recognition sequence of M.TaqI was employed. Nine of ten phages selected were found to have the same deduced amino acid sequence SVSVGMKPSPRP. The selected phage binds to DNA, as demonstrated in an ELISA.     

Amyloid-forming peptides selected proteolytically from phage display library

We demonstrated that amyloid-forming peptides could be selected from phage-displayed library via proteolysis-based selection protocol. The library of 28-residue peptides based on a sequence of the second zinc finger domain of Zif268, and computationally designed betabetaalpha peptide, FSD-1, was presented monovalently on the surface of M13 phage. The library coupled the infectivity of phage particles to proteolytic stability of a peptide introduced into the coat protein III linker. It was designed to include variants with a strong potential to fold into betabetaalpha motif of zinc finger domains, as expected from secondary structure propensities, but with no structure stabilization via zinc ion coordination. As our primary goal was to find novel monomeric betabetaalpha peptides, the library was selected for stable domains with the assumption that folded proteins are resistant to proteolysis. After less than four rounds of proteolytic selection with trypsin, chymotrypsin, or proteinase K, we obtained a number of proteolysis-resistant phage clones containing several potential sites for proteolytic attack with the proteinases. Eight peptides showing the highest proteolysis resistance were expressed and purified in a phage-free form. When characterized, the peptides possessed proteolytic resistance largely exceeding that of the second zinc finger domain of Zif268 and FSD-1. Six of the characterized peptides formed fibrils when solubilized at high concentrations. Three of them assembled into amyloids as determined through CD measurements, Congo red and thioflavin T binding, and transmission electron microscopy.     

Peptides selected from phage display library may change the conformation of S protein of rice stripe virus

Summary Phages with high affinity to the S protein obtained from rice stripe virus (RSV) were enriched from phage-displayed random 12-mer peptide library after three rounds of biopanning. 9 different peptides from the enriched library were selected by ELISA. Circular dichroism (CD) spectra of the GST-S fusion protein with binding phages and non-binding phages showed that structure of the S protein was changed after it bound to each of these 9 selected 12-mer peptides, which suggested that these peptides might disrupt the function of S protein. Thus, those peptides might be used to develop plant resistance and disrupt virus transmission. 3 of the 12-mer peptide genes were fused with the GST gene in pGEX 3X. The fusion proteins were also obtained usingE. coli expression system and purified.     

Peptides selected from phage display library may change the conformation of S protein of rice stripe virus

Phages with high affinity to the S protein obtained fromrice stripe virus (RSV) were enriched fromphage-displayed random 12-mer peptide library after threerounds of biopanning. 9 different peptides from theenriched library were selected by ELISA. Circulardichroism (CD) spectra of the GST-S fusion protein withbinding phages and non-binding phages showed thatstructure of the S protein was changed after it bound toeach of these 9 selected 12-mer peptides, which suggestedthat these peptides might disrupt the function of Sprotein. Thus, those peptides might be used to developplant resistance and disrupt virus transmission. 3 of the12-mer peptide genes were fused with the GST gene in pGEX3X. The fusion proteins were also obtained using E.coli expression system and purified.     

噬菌体抗体库的优化

噬菌体抗体组合文库技术作为噬菌体展示和抗体组合文库两种技术的集成,由于它具有库容量大、特异性高、和敏感性强的优点而被誉为抗体技术的第三次革命。但是由于一些技术上的原因,使得它无法得到广泛的应用,本文就其优化进行综述。     

Identification of the amino acid-AZT-phosphoramidase by affinity T7 phage display selection

A CEM cell cDNA T7 phage display library was prepared and used to screen for activating enzymes of phosphoramidate prodrugs of AZT monophosphate. Although, inefficient compared to ribonucleotide based phosphoramidates, hHint 1 was identified as the likely intracellular pronucleotide activating enzyme.     

Construction and diversity analysis of a murine IgE phage surface display library

INTR0DUCTI0NIn0urprevi0usstudy0fV5andVKfromfouranti-TCSIgEhybrid0masestab-lishedin0urlaboratory,wefoundVKfr0mallfourcl0nesusedfragmentsfromthesamegermlinegenefamilyVK21,andabiasintheuse0fJk1genefragmentwasalsoobserved.Ontheotherhand,thegeneusageofVHwaJsquitediverse.WespeculatedthatinIgEresponsest0TCS,thelightchainmayplayam0reimpor-tantroleinspecificbindingtoallergenicdeterminant0nTCS[1].However,duet0thelimitationofhybrid0matechnology,itisdifficulttoachievealargenumberofanti-TCSI…     

Specific glycosidase activity isolated from a random phage display antibody library

Carbohydrates serve as key receptor sites in various cellular events such as viral attachment, tumor formation, and tissue inflammation. A potential route to control these events is to manipulate targeted carbohydrate structures in vivo using specifically designed glycohydrolases. Here we show that a stereospecific catalytic activity designed toward a particular sugar and linkage can be readily isolated from a phage display antibody library derived from a nonimmunized host. The activity was isolated using a transition-state analogue mimicking an alpha-glucosidasic linkage as antigen and showed a 20-fold specificity for that sugar and linkage. The DNA sequence, however, contains a large deletion in the antibody gene, which also changes the downstream reading frame, resulting in a translated sequence containing only 57 amino acids that has a predominantly hydrophobic amino terminal and a strongly hydrophilic carboxy terminal. The isolated catalytic activity has a strong pH dependence, attributable to one or more of the numerous potentially charged groups in the carboxyl terminal. While the protein readily forms more stable multimers, the 7.3-kD monomer represents by far the smallest glycosidase enzyme reported to date and can provide substantial new information toward understanding and modifying glycosidase activity.     

Identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a QCM device

Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a self-assembled monolayer (SAM) was synthesized and used for immobilization on the gold electrode surface of a QCM sensor chip. After six sets of one-cycle affinity selection, T7 phage particles displaying PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-mer) were found to be enriched at a rate of 17/44, 9/44, respectively, suggesting that these peptides specifically recognize trimannoside. Binding checks using the respective single T7 phage and synthetic peptide also confirmed the specific binding of these sequences to the trimannoside-SAM. Subsequent analysis revealed that these sequences correspond to part of the primary amino acid sequence found in many mannose- or hexose-related proteins. Taken together, these results demonstrate the effectiveness of our T7 phage display environment for affinity selection of binding peptides. We anticipate this screening result will also be extremely useful in the development of inhibitors or drug delivery systems targeting polysaccharides as well as further investigations into the function of carbohydrates in vivo.