核糖体展示单链抗体库技术及应用的研究进展

核糖体展示是一种完全离体进行的功能蛋白筛选和鉴定的新技术,避免了传统的体内筛选技术的缺陷,使得文库容量增大、分子多样性加强。它将正确折叠的蛋白及其mRNA同时结合在核糖体上,形成mRNA-核糖体-蛋白质三聚体,使目的蛋白的基因型和表型联系起来。近年来,核糖体展示技术在单链抗体(ScFv)库构建及应用方面取得了很大进展。现就这方面的研究进展作一综述。     

蛋白质体外表达与进化技术

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。     

核糖体展示技术的研究与应用现状

核糖体展示技术是20世纪90年代建立并发展起来的一种完全在体外进行的分子筛选与进化技术,避免了体内展示技术的缺陷,不受细胞转染与基因表达等因素影响,已经成功用于筛选与靶分子特异结合的高亲和力多肽、抗体和酶等.本文主要概述了核糖体展示技术的基本原理、具体步骤、技术改进及应用现状.     

核糖体展示口蹄疫单链抗体库的构建

目的:构建库容量大、多样性好的核糖体展示口蹄疫单链抗体(scFv)库。方法: 分离口蹄疫病毒免疫的兔脾细胞,提取总RNA,用RT-PCR扩增兔抗体的重链可变区(VH)基因和轻链可变区(VL)基因,同时扩增作为间隔区的兔抗体Ck基因;采用重叠延伸PCR (简称SOE-PCR)技术连接VH-VL基因,同时引入T7启动子和核糖体结合位点序列,体外构建核糖体展示scFv库模板,连接pMD18-T载体转化E.coli DH5α大肠杆菌,挑取阳性克隆测序以鉴定scFv组装。结果:成功构建了库容量达8.21×10¹³的兔源口蹄疫核糖体展示scFv库。结论: 构建的大容量兔源性口蹄疫核糖体展示抗体库可以成为进一步筛选特异性口蹄疫单链抗体的实验平台,为开发诊断性口蹄疫单链抗体奠定了很好的实验基础。     

利用核糖体展示技术筛选口蹄疫病毒 单链抗体基因

目的:利用核糖体展示技术筛选口蹄疫病毒特异性单链抗体基因。方法:在已构建好的核糖体展示文库的基础上,利用核糖体展示技术,经过5轮的体外转录、体外转译、亲和筛选和RT-PCR,将得到的序列进行测序分析。结果:筛选到FMDV scFv基因,且基因得到富集。结论:实验运用核糖体展示技术,以FMDV抗原和纯化的146S病毒粒子为靶标筛选到了FMDV scFv基因,将为scFv用于FMD的基础研究、免疫学研究以及为预防、治疗和诊断提供帮助,也为研制FMD的快速诊断技术奠定先前基础。     

一种核糖体展示单链抗体文库的构建与鉴定

用柑桔溃疡病致病菌Xanthomonas axonopodis pv. citri（Xac）全菌免疫小鼠,提取小鼠脾细胞mRNA,RT-PCR扩增小鼠抗体重链可变区（VH）和轻链可变区（VL）,采用linker (Gly4Ser)3连接VH和VL,构建用于核糖体展示方法筛选阳性单链抗体（scFvs）的文库。通过将scFv文库DNA转化大肠杆菌JM109,随机挑取克隆子测序以分析单链抗体文库的多样性。结果显示,用柑桔溃疡病菌免疫后的小鼠抗血清效价为2500倍左右,扩增的VH大小为350bp左右,VL的大小为650bp左右,linker连接后的单链抗体文库DNA大小为1200bp左右。测序结果显示,单链抗体文库多样性好。以Xac为靶,筛选到了抗Xac的特异性抗体,说明该抗体库可用于柑桔溃疡病菌单链抗体的筛选。     

核糖体展示技术

组合化学在分子生物学中的应用,大大提高了所构建分子文库的容量,而高通量筛选技术对于大容量文库的筛选是至关重要的。核糖体展示技术克服了传统筛选技术库容量小和筛选效率低等缺陷。该技术完全在体外进行,其文库容量不受细胞转染效率的影响,库容量和筛选效率得到很大提高,可与其他技术相组合,在体外分子高效表达和定向进化方面具有广泛的应用前景。该文介绍核糖体展示技术的基本原理、技术特点以及最新研究进展。     

基于核糖体展示技术进行抗狂犬病毒人源抗体的制备

构建了核糖体展示人源抗狂犬病毒单链抗体(scFv)库,筛选制备特异抗狂犬病毒糖蛋白(RVGp)的稳定性人源抗体.应用核糖体抗体库技术,从经狂犬病毒Vero疫苗免疫的志愿者外周血淋巴细胞中分离、构建核糖体展示scFv基因库.体外转录翻译后,以RVGp重组蛋白作筛选抗原,采用亲和富集法淘选RVGp特异性scFv抗体基因.在原核系统pET22b(+)/BL21(DE3)中实现scFv抗体片段的可溶性表达,ELISA鉴定阳性克隆.然后对筛选的scFv进行稳定性改构,构建VH-Lc-VK稳定性抗体,并对其生物学活性进行初步研究.成功构建了库容量约为6.2×1012的核糖体展示scFv抗体基因库.在180个筛选克隆中,克隆RB24、RB71、RB109和RB156显示出较高的ELISA值,其基因序列分析结果显示,它们是全新的人源抗RVGp抗体.改构后的抗RVGp VH-Lc-VK抗体的稳定性明显改进,可特异识别RVGp并有效中和狂犬病毒,抑制狂犬病毒对靶细胞的感染.以上结果表明,人源抗RVGp特异性抗体的获得,为狂犬病的有效预防、诊断和治疗提供了新的途径,而且将为其他人源抗体的制备提供理论依据和技术基础.     

核糖体展示研究进展

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。     

核糖体展示及体外分子选择与进化

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。     

Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target

Ribosome display is an in vitro selection and evolution technology for proteins and peptides from large libraries. As it is performed entirely in vitro, there are two main advantages over other selection technologies. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be introduced easily after each selection round, as no library must be transformed after any diversification step. This allows facile directed evolution of binding proteins over several generations. A prerequisite for the selection of proteins from libraries is the coupling of genotype (RNA, DNA) and phenotype (protein). In ribosome display, this link is accomplished during in vitro translation by stabilizing the complex consisting of the ribosome, the mRNA and the nascent, correctly folded polypeptide. The DNA library coding for a particular library of binding proteins is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. The ribosomal complexes are allowed to bind to surface-immobilized target. Whereas non-bound complexes are washed away, mRNA of the complexes displaying a binding polypeptide can be recovered, and thus, the genetic information of the binding polypeptides is available for analysis. Here we describe a step-by-step procedure to perform ribosome display selection using an Escherichia coli S30 extract for in vitro translation, based on the work originally described and further refined in our laboratory. A protocol that makes use of eukaryotic in vitro translation systems for ribosome display is also included in this issue.     

mRNA display: ligand discovery,interaction analysis and beyond

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.     

抗脐带间充质干细胞表面分子噬菌体ScFv抗体库的构建及初步鉴定

目的：利用噬菌体展示技术构建抗脐带间充质干细胞表面分子噬菌体ScFv抗体库。方法：收集P3代培养的UC-MSCs免疫BALB/c小鼠,提取其脾细胞总RNA,RT-PCR扩增全套VH和VL基因片段,将其先后克隆入噬菌粒pSEX81中,构建成完整的噬菌体ScFv抗体库。结果:构建的噬菌体ScFv抗体库的库容为2×107cfu,ScFv插入重组率为93％,BstN1酶切图谱呈不同多样性。ScFv抗体库经3轮初步筛选后插入重组率达100％,3个克隆出现了相同的酶切图谱,并且随着筛选次数的增加,输出/输入比明显提高,这说明抗体库得到了特异性富集。结论：成功地构建了抗脐带间充质干细胞表面分子噬菌体ScFv抗体库,这为将来筛选特异性抗体和进一步用于间充质干细胞表面特异性分子研究奠定了坚实的基础。     

人源天然ScFv噬菌体抗体库的构建及鉴定

噬菌体抗体库技术是获得治疗性抗体的一条重要途径。以20份健康人外周血为样本,通过提取淋巴细胞、逆转录－PCR（RT PCR）、抗体可变区基因的扩增、重叠PCR获得单链抗体（ScFv）基因,将ScFv克隆入噬粒载体,通过近300次的电转化获得了库容量为1.3×109的全人源天然ScFv噬菌体抗体库。通过随机挑克隆测序和用5种不同抗原筛选对抗体库进行了初步验证。随机测序表明抗体库具有较好的多样性,用5种不同抗原对其进行筛选,均获得了特异性噬菌体抗体的不同富集,表明成功构建了一个多样性良好的人源天然ScFv噬菌体抗体库。     

Antibody evolution from the centre to the periphery: applied to a human antibody fragment recognising the tumour-associated antigen mucin-1

Mucin-1 has proven to be a suitable target for antibody-based diagnosis and therapy of certain tumours, but no appropriate human antibody or antibody fragment displaying slow dissociation rate kinetics against this target is available. Since a rapid dissociation character prevents an antibody fragment from remaining at the site of the antigen, this fact may prevent the successful application of a human mucin-1 specific antibody in diagnosis and therapy. We have now used iterative antibody libraries to evolve a human antibody fragment originally obtained from a na?ve antibody library. A strategy was devised whereby molecular variants displaying slow dissociation kinetics against the repetitive mucin-1 tumour-associated antigen can be selected in vitro. The evolved clones, that allowed for a reduced dissociation from the tumour antigen, carried substitutions in the outer parts of the binding site. This demonstrated the ability of this in vitro evolution technique to mimic the process whereby antibodies evolve in vivo. We have thus devised a strategy through which molecular variants displaying slow dissociation from a repetitive target like the mucin-1 tumour-associated antigen can be obtained in vitro. These or related molecules obtained by this approach will serve as a starting point for the development of fully human antibodies for use in mucin-1 specific tumour therapy of diagnosis.     

噬菌体抗体库技术与高通量筛选抗体

噬菌体抗体库技术是组合技术与基因工程抗体技术相结合的产物 ,为快速筛选特异性抗体提供了简便而高效的操作系统 ,随着蛋白质组学的飞速发展 ,对抗体的大规模制备的需求日益增加 ,迫切需要发展高质量的抗体库和与之相整合的高通量筛选技术。近年来 ,以上技术的发展和自动化设备的引入为大规模抗体制备的实现提供了条件 ,对这一领域的研究进展做一概述。     

DNA display II. Genetic manipulation of combinatorial chemistry libraries for small-molecule evolution

Biological in vitro selection techniques, such as RNA aptamer methods and mRNA display, have proven to be powerful approaches for engineering molecules with novel functions. These techniques are based on iterative amplification of biopolymer libraries, interposed by selection for a desired functional property. Rare, promising compounds are enriched over multiple generations of a constantly replicating molecular population, and subsequently identified. The restriction of such methods to DNA, RNA, and polypeptides precludes their use for small-molecule discovery. To overcome this limitation, we have directed the synthesis of combinatorial chemistry libraries with DNA "genes," making possible iterative amplification of a nonbiological molecular species. By differential hybridization during the course of a traditional split-and-pool combinatorial synthesis, the DNA sequence of each gene is read out and translated into a unique small-molecule structure. This "chemical translation" provides practical access to synthetic compound populations 1 million-fold more complex than state-of-the-art combinatorial libraries. We carried out an in vitro selection experiment (iterated chemical translation, selection, and amplification) on a library of 10(6) nonnatural peptides. The library converged over three generations to a high-affinity protein ligand. The ability to genetically encode diverse classes of synthetic transformations enables the in vitro selection and potential evolution of an essentially limitless collection of compound families, opening new avenues to drug discovery, catalyst design, and the development of a materials science "biology."     

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。     

Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display

Here we applied ribosome display to in vitro selection and evolution of single-chain antibody fragments (scFvs) from a large synthetic library (Human Combinatorial Antibody Library; HuCAL) against bovine insulin. In three independent ribosome display experiments different clusters of closely related scFvs were selected, all of which bound the antigen with high affinity and specificity. All selected scFvs had affinity-matured up to 40-fold compared to their HuCAL progenitors, by accumulating point mutations during the ribosome display cycles. The dissociation constants of the isolated scFvs were as low as 82 pM, which validates the design of the na?ve library and the power of this evolutionary method. We have thus mimicked the process of antibody generation and affinity maturation with a synthetic library in a cell-free system in just a few days, obtaining molecules with higher affinities than most natural antibodies.     

Photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins

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.