噬菌体表面呈现技术在肿瘤中的应用

噬菌体表面呈现技术（Phage display technology)是近年来发展起来的新兴技术,目前将噬菌体表面呈现技术用于肿瘤,在肿瘤治疗方面取得了很大的进展,本文就噬菌体表面呈现技术在肿瘤研究中的应用与前景加以综述。     

丝状噬菌体表面呈现技术的进展

本文围绕建库和扩库、筛选和检测,探讨了噬菌体表面呈现技术的发展。一些新的设计是对经典方法的有益补充。相似的表面呈现系统正在被开发。     

噬菌体呈现肽库技术的应用

噬菌体呈现肽库是噬菌体显示技术的一个非常重要的分支。自问世以来,随着分子生物学技术的飞速发展,它已被广泛应用于免疫学、分子生物学、药理学、疫苗学等生命科学领域。简要概述了这一技术的应用。     

新型噬菌体表面呈现载体的构建

作为抗体库筛选的一个有效方法,噬菌体表面呈现技术在单链抗体的研制和中得到广泛的应用。以噬菌粒pCANTAB5E和pHB为基础。利用PCR和DNA重组方法,构建了一个新型的用于单链抗体噬菌体表面呈现的噬菌粒载体,随后用一株对大肠杆菌细胞有毒性的人源化单链抗体（1HSCFV）对其呈现单链抗体的效果进行了初步的评价。结果表明新型噬菌粒系统具有更好的呈现能力。     

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].     

丝状噬菌体表面呈现技术

对丝状噬菌体的结构、基因组及生命周期的深入认识,是丝状噬菌体表面呈现技术建立和发展的基础,可将外源基因片段插入噬菌体的基因Ⅲ（ｇ３）或基因Ⅷ（ｇ８）的先导序列的紧下游,使外源基因表达的多肽以融合蛋白的形式呈现有噬菌体表面外壳蛋白ｇｐ３或ｇｐ８的Ｎ端,这样的呈现常能使表达的多肽保持生物活笥,据此可用活的噬菌体直接方便,高效率地筛选目的基因或检测该基因产物的活性,这技术已被用于抗体基因库,ＣＤＮＡ     

细菌表面呈现技术研究进展

自从首次描述外源蛋白在大肠杆菌表面呈现成功以来,细菌表面呈现技术得到了迅猛的发展,无论是革兰氏阴性菌还是革兰氏阳性菌都可用于异源蛋白的表面呈现,该技术被应用于微生物学、免疫学、分子生物学、疫苗学以及生物工程的多个领域的基础和应用研究。     

噬菌体呈现技术制备结肠癌抗独特型抗体

 以纯化的鼠抗人结肠癌细胞单克隆抗体 (简称单抗 )MC5与钥孔血蓝素 (KLH)的交联物经腹腔免疫Balb c小鼠 ,取脾分离mRNA .RT PCR分别扩增抗体重、轻链可变区基因片段 (VH 和VLcD NA ,大小分别约为 340bp和 32 0bp) ,二者经linkerDNA连接形成ScFv(singlechainvariablefragment)DNA(约 75 0bp) .将ScFvDNA与噬菌粒载体pCANTAB5E的连接产物转化于大肠杆菌TG1,经辅助噬菌体M13KO7感染后 ,获得重组噬菌体抗体ScFv文库 .以单抗MC5对ScFv文库进行 4轮亲和筛选后 ,随机挑取 80个克隆经酶联免疫吸附实验 (ELISA)筛选出 2 2个呈现ScFv形式抗独特型抗体(抗 IdScFv)的噬菌体单克隆 .竞争抑制实验表明 ,在 2 2个阳性克隆中有 4个克隆所呈现的抗 IdScFv属β或γ型 .针对单抗MC5的噬菌体呈现型抗 IdScFv的制备 ,为筛选新的结肠癌重组抗 Id瘤苗候选分子奠定了基础     

噬菌体6肽随机表面表达文库的构建

体外合成编码６肽的随机ＤＮＡ片段以及用于随机ＤＮＡ片段扩增了一对ＰＣＲ引物,再经ＰＣＲ扩增,ＢｇＩＩ酶切扩增产物,获得编码６肽的随机ＤＮＡ克隆片段,并利用已构建的噬菌体表面表达载体,经抗性和插入复活筛选,获得１．６×１０＾８个独立克隆,构成库的克隆经酶切,ＰＣＲ扩增,斑点杂交,序列测定,亲和素富集等方法的综合鉴定和评定,以及６肽随机克隆体外增殖和表达特性观察,结果均表明,我们成功构建了编码６肽的     

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。     

噬菌体展示技术系统发展进展

噬菌体展示技术(Phage display technology,PDT)是一种特殊的基因工程重组表达技术,噬菌体展示技术系统(Phage display system,PDS)是指包括经过遗传改造后的系列噬菌体、辅助噬菌体、宿主细菌等集成平台(含试剂盒)。文章从噬菌体分子遗传学及其基因(基因组)遗传工程改良角度,基于噬菌体M13、λ、T4和T7等4大类典型噬菌体展示技术系统的发展进展进行了综述。重点强调不同展示系统中的核心部件及其基因工程改造的分子遗传学原理、不同展示锚定位点的技术特征、相关试剂盒的研制状况及选择依据。     

Efficient identification of phosphatidylserine-binding proteins by ORF phage display

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in ∼300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.     

Engineering high affinity superantigens by phage display

Protein L (PpL) is a B-cell superantigen from Peptostreptococcus magnus known to bind to mammalian Vkappa light chains. PpL from P.magnus strain 312 comprises five homologous immunoglobulin (Ig) binding domains. We first analysed the binding of the individual domains (B1-B5) of PpL(312) to human Vkappa light chains (huVkappa) subtypes 1 (huVkappaI) and 3 (huVkappaIII). Using a combination of rational design and phage selection we isolated mutants of the N-terminal B1 domain with a 14-fold increased affinity for huVkappa1 (B1kappa1) and >tenfold increased affinity for huVkappaIII (B1kappa3). We investigated the potential of the selected domains, in particular the B1kappa1 domain, as reagents in immunochemistry and immunotherapy. B1kappa1 proved a superior reagent than the wild-type domain, allowing up to tenfold more sensitive detection of human Vkappa antibody fragments in ELISA. A fusion protein of B1kappa1 with a human Vlambda antibody scFv fragment promoted the efficient recruitment of antibody encoded effector functions including complement, mononuclear phagocyte respiratory burst and phagocytosis through retargeting of IgGkappa and IgMkappa. Our results suggest that superantigens with improved affinity and/or specificity are easily accessible through protein engineering. Such engineered superantigens should prove useful as reagents in immunochemistry and may have potential as agents in immunotherapy.     

A phage display system for studying the sequence determinants of protein folding.

We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.     

Concepts in antibody phage display.

This paper introduces the reader to antibody phage display and its use in combinatorial biochemistry. The focus is on overviewing phage display formats, library design and selection technology, which are the prerequisites for the successful isolation of specific antibody fragments against a diverse set of target antigens.     

噬菌体展示库筛选细胞特异分子的进展

介绍噬菌体展示技术的原理和发展,尤其是噬菌体展示技术在筛选细胞特异分子的策略方面的进展。该技术通过20年的发展已成为一种研究抗原一抗体作用、蛋白质相互作用、蛋白一药物相互作用甚至蛋白质一核酸作用的分析手段,但涉及到以完整细胞、器官或组织等复杂的生物活性分子表面为靶标则筛选效果尚不理想。关键是要减少噬菌体展示分子与靶标的非特异性结合,利用更为严格的经过改进的筛选策略。该技术的优势预示着它将广泛被应用于基础理论和研究实践中。     

Corruption of phage display libraries by target-unrelated clones: Diagnosis and countermeasures

Phage display is used to discover peptides or proteins with a desired target property—most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phages or peptides (TUPs), that lack the target behavior. Many TUPs are propagation related; they have mutations conferring a growth advantage and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus-strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus-strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus-strand origin. The founder’s infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption.     

Novel p16 binding peptide development for p16‐overexpressing cancer cell detection using phage display

Protein p^16INK4a (p16) is a well‐known biomarker for diagnosis of human papillomavirus (HPV) related cancers. In this work, we identify novel p16 binding peptides by using phage display selection method. A random heptamer phage display library was screened on purified recombinant p16 protein‐coated plates to elute only the bound phages from p16 surfaces. Binding affinity of the bound phages was compared with each other by enzyme‐linked immunosorbent assay (ELISA), fluorescence imaging technique, and bioinformatic computations. Binding specificity and binding selectivity of the best candidate phage‐displayed p16 binding peptide were evaluated by peptide blocking experiment in competition with p16 monoclonal antibody and fluorescence imaging technique, respectively. Five candidate phage‐displayed peptides were isolated from the phage display selection method. All candidate p16 binding phages show better binding affinity than wild‐type phage in ELISA test, but only three of them can discriminate p16‐overexpressing cancer cell, CaSki, from normal uterine fibroblast cell, HUF, with relative fluorescence intensities from 2.6 to 4.2‐fold greater than those of wild‐type phage. Bioinformatic results indicate that peptide ‘Ser‐His‐Ser‐Leu‐Leu‐Ser‐Ser’ binds to p16 molecule with the best binding score and does not interfere with the common protein functions of p16. Peptide blocking experiment shows that the phage‐displayed peptide ‘Ser‐His‐Ser‐Leu‐Leu‐Ser‐Ser’ can conceal p16 from monoclonal antibody interaction. This phage clone also selectively interacts with the p16 positive cell lines, and thus, it can be applied for p16‐overexpressing cell detection. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.