噬菌体展示技术及其在血吸虫研究中的应用

血吸虫是严重危害人类健康的寄生虫之一,近几年噬菌体展示技术为血吸虫病防治研究提供了新的思路和方法,在血吸虫病的诊断、治疗、预防中起到一定的作用,现综述如下.     

利用噬菌体载体展示抗体文库技术研究进展

1985年,美国Missouri大学的SmithGP将外源基因插入丝状噬菌体（Filamentous bacteriophage,fd）的基因组,使目的基因编码的多肽以融合蛋白的形式展示,从而创建了噬菌体展示技术。1994年McCaffery等最先构建了噬菌体抗体展示文库,开始了噬菌体展示技术应用的新时代。十多年来,噬菌体展示技术被用于蛋白质、多肽（如抗体、酶）的制备,主要应用于医学、生物、食品等领域。     

噬菌体抗体库技术研究进展

噬菌体展示技术（PDT）在模拟表位的筛选、筛选特定靶分子的配体、蛋白与蛋白间相互作用、细胞信号转导等基础研究，疾病的治疗和致病机理研究以及疫苗的制备、基因工程抗体的研发等领域均有广泛应用。噬菌体抗体库技术是通过PCR将全套人抗体重链和轻链V区基因克隆出来，并在噬菌体表面表达、分泌，经过筛选后获得特异性抗体的技术。该技术融合了丝状噬菌体展示与抗体组合文库技术，得到的抗体称为噬菌体抗体。     

噬菌体展示技术筛选乙型肝炎病毒核心启动子结合蛋白的研究

目的：通过筛选乙型肝炎病毒(HBV)核心启动子(core promoter，CP)结合蛋白，为HBV复制机制和调控机制的研究探索新的途径。方法：应用噬菌体展示技术，以HBV的核心启动子的聚合酶链反应(PCR)产物作为固相筛选分子，对噬菌体人肝细胞cDNA文库进行4轮“吸附-洗脱-扩增”筛选过程，经噬斑的PCR扩增后。构建克隆载体，最后对所筛选克隆进行DNA序列分析和同源性搜索。结果：噬菌体经富集后，随机筛选得到7个阳性克隆．成功构建了克隆载体。序列测定后经过同源性搜索，确定了和HBV启动子结合的肝细胞蛋白基因序列，分别编码β2微球蛋白和3种未知功能蛋白。结论：用噬菌体人肝cDNA文库筛选得到了与HBV核心启动子结合的蛋白，分析了该蛋白的编码基因，为进一步研究HBV与肝细胞相互作用的分子生物学机制开辟了新的途径。     

应用噬菌体展示技术筛选原发性肝癌血清结合蛋白

目的:通过筛选原发性肝癌血清蛋白结合蛋白,探究肝癌的分子发病机制.方法:应用噬菌体展示技术,以肝癌患者的血清作为固相筛选分子,对T7 Select噬菌体人肝细胞cDNA文库进行5轮"吸附-洗脱-扩增"的筛选过程,经噬斑的聚合酶链式反应(PCR)扩增后,对阳性PCR产物直接进行序列测定和同源性分析.结果:噬菌体经富集后,随机挑取48个噬斑进行PCR扩增,得到5种大小不同的PCR片断,序列测定后经序列同源性分析,结果发现与肝癌患者血清蛋白结合的蛋白有以下5种:人核仁螺旋体磷酸化蛋白NOLC1,人高度迁移基核小体结合域1(HMGN1),人依赖ATP的DNA连接酶1(LIG1),人小EDRK-丰富因子2(SERF2)和A-kinase anchor protein 9 isoform.结论:用噬菌体人肝cDNA文库筛选得到了肝癌血清蛋白结合蛋白,为进一步阐明肝癌的发生及发病机制奠定了基础.     

噬菌体展示技术在鼠CD137L活性研究中的应用

目的探讨噬菌体展示技术在鼠CD 137配体(CD 137L)活性研究中的应用。方法用PCR法扩增出鼠CD 137L胞外区,用噬菌体展示技术展示其CD 137L;EL ISA方法检测噬菌体CD 137L的抗原性,并分3次注射到大鼠体内,取血清检测大鼠体内多克隆抗体水平。结果PCR方法成功扩增出鼠CD 137L胞外区;获得重组子pCom b3H-CD 137L,成功展示在噬菌体上;噬菌体CD 137L可与抗CD 137L特异性结合;大鼠体内产生了CD 137L的多克隆抗体。结论噬菌体展示技术用于鼠CD 137L活性的研究,可提高制备特异性抗体的成功率,得到特定结构和功能的蛋白质。     

噬菌体展示技术及其在弓形虫研究中的应用

将病原体的免疫原与λ噬菌体或其他病毒的衣壳蛋白融合后一起展示于病毒表面 ,这样形成的新的病毒颗粒可用作疫苗。Smith[1] 通过基因工程手段将外源基因插入到丝状噬菌体的PIII蛋白基因中 ,成功地使外源多肽展示在噬菌体的表面 ,而且用抗体检测发现该外源多肽具有同天然产物相同的生物活性。这标志着噬菌体展示技术的诞生。而Geysen等[2 ] 认为含有关键残基的短肽能够模拟蛋白质上的功能位点 ,而且多数情况下 ,几个关键残基与其受体所形成的非共价键构成了全部结合的主要部分。这就为噬菌体展示技术奠定了理论基础。1　噬菌体展示技术噬…     

噬菌体展示技术筛选乙型肝炎病毒增强子Ⅰ结合蛋白的研究

目的　通过筛选乙型肝炎病毒 (HBV)增强子Ⅰ (Enhl)结合蛋白 ,为HBV复制机制的研究探索新的途径。方法　应用噬菌体展示技术 ,以HBV的增强子Ⅰ的聚合酶链反应 (PCR)产物DNA作为固相筛选分子 ,对噬菌体人肝细胞cDNA文库进行 4轮“吸附 洗脱 扩增”筛选过程 ,经噬斑的PCR扩增后 ,构建克隆载体 ,最后对所筛选克隆进行DNA序列分析和同源性搜索。结果　噬菌体经富集后 ,从随机筛选的 12个克隆中得到 6个阳性克隆 ,成功构建了克隆载体。序列测定后经过同源性搜索 ,确定了和HBV增强子Ⅰ结合的肝细胞蛋白 ,共编码 3种蛋白。分别为 3 磷脂酰肌醇激酶相关蛋白激酶、人类血清白蛋白、核膜孔复合体相互作用蛋白。结论　用噬菌体人肝cDNA文库筛选得到了与HBV增强子Ⅰ结合的蛋白 ,分析了该蛋白的编码基因 ,可能是作用于增强子Ⅰ的反式作用因子 ,但它们对HBV增强子Ⅰ是否有转录调控作用还需进行进一步的研究证实。     

噬菌体表面展示技术筛选隐源性肝炎血清蛋白结合蛋白

目的:通过筛选隐源性肝炎血清蛋白结合蛋白,为隐源性肝炎的发病机制研究探索新途径.方法:应用噬菌体表面展示技术,将隐源性肝炎患者血清作为固相筛选蛋白,用噬菌体正常人肝细胞T7cDNA文库进行5轮生物筛选,经噬斑PCR,对筛选克隆进行DNA序列分析,将推断的氨基酸序列在蛋白质库中进行搜索,自GenBank中获得结合蛋白的cDNA和基因组的全长序列,然后再探讨该蛋白在隐源性肝炎发病机制中的作用.结果:噬菌体经过5轮生物富集后,从随机筛选的60个克隆中得到24个阳性克隆,经序列测定后进行同源搜索,初步确定人类HCVNS5A转化调节蛋白13(NS5ATP13)为人体肝脏中固有的与隐源性肝炎血清蛋白结合的蛋白.结论:应用T7cDNA噬菌体表面展示技术,我们发现隐源性肝炎血清蛋白与人类肝细胞HCVNS5A转化调节蛋白13相互作用,在隐源性肝炎发生及抗病毒治疗有重要意义.     

噬菌体展示布鲁氏菌蛋白文库构建及差减筛选融合蛋白

目的 建立噬菌体展示羊型布鲁氏菌16M株表面蛋白文库,差减筛选具有良好特异性、亲和力及反应原性的蛋白,为确定新型诊断用抗原奠定基础。方法 利用噬菌粒载体pYW01构建重组羊型布鲁氏菌16M株基因文库,辅助噬菌体VCSM13感染基因文库进而构建噬菌体展示羊型布鲁氏菌16M蛋白文库。利用PEG纯化后,扩增蛋白文库。随机挑取单克隆,提取质粒,测序鉴定蛋白文库。通过差减筛选,选择具有良好特异性、亲和力及反应原性的表面蛋白,为确定诊断用抗原奠定基础。结果 通过DNA重组技术,构建羊型布鲁氏菌16M基因文库,库容量达到10⁸ pfu/ mL,并且随机性良好。利用辅助噬菌体VCSM13包装基因文库,随机挑取蛋白文库中单克隆,提取质粒,经测序鉴定,成功构建噬菌体展示羊型布鲁氏菌16M株表面蛋白文库。利用免疫血清及感染血清对噬菌体展示蛋白文库进行差减淘选,筛选出6个噬菌体融合蛋白。经Western-blot分析6个噬菌体融合蛋白均具有较好的反应原性及特异性。结论 成功构建噬菌体展示布鲁氏菌蛋白文库,差减筛选出6个具有较好反应原性及特异性的融合蛋白,为后续新型血清学诊断制剂筛选奠定基础。     

噬菌体展示技术的应用及研究进展

噬菌体展示技术是将外源蛋白或多肽与噬菌体外壳蛋白融合并呈现于噬菌体表面,利用外源蛋白或多肽与筛选靶标的特异性亲和作用,通过吸附、洗脱、扩增的重复过程,将含有特异性外源蛋白的噬菌体从表达有各种外源蛋白的噬菌体库中筛选出来,并得到大量富集,而后进行基因序列测定。1985年,Smith[1]通过基因工程手段将外源蛋白质与噬菌体的次要外壳蛋白pⅢ形成融合蛋白展示于噬菌体颗粒表面,可保持相对独立的空间构象和生物活性,而不影响重组噬菌体对宿主菌的感染能力。与其他表达系统相比噬菌体展示技术具有显著的优点:可将基因型和表型、分子结…     

Applying phage display technology in aging research

Filamentous bacteriophage offers the possibility oflinking genotype with phenotype in one geneticpackage. By creating a library of different proteinsas fusion to one of the coat proteins of filamentousbacteriophage it becomes possible to isolate proteinsbased on their binding characteristics. Phagedisplayed libraries of varying formats, ranging fromsmall peptides to antibody fragments, have beenselected successfully for ligands toward a large panelof targets. These peptides and antibody fragments areapplicable in analysis of the behaviour of certainproteins with respect to changes in biologicalprocesses such as aging, thus providing valuable toolsfor a general understanding of the aging process.Alternatively, display of antibody fragments orpeptides on filamentous bacteriophage can beinstrumental in the discovery of novel antigens asexemplified by selections on cell surfaces or oncomplex protein mixtures such as sera from agingindividuals. Although phage display has been appliedsuccessfully in a large number of studies relating tocancer, viral infections and other biologicalprocesses, its application in the field of agingresearch is yet to be realised.     

噬菌体展示技术在结核病研究中的应用

随着艾滋病患者的增加和MTB耐药菌株的流行与传播,使结核病的诊断和治疗面临更大的挑战.近年来迅速发展的一种体外分子进化技术-噬菌体展示技术在结核病研究中呈现出独特的优势.     

噬菌体随机肽库技术及其在寄生虫学领域的应用

198 5年Smith率先将EcoRI内切酶的部分基因片段插入丝状噬菌体fl的外壳蛋白基因Ⅲ区 ,使目的基因编码的多肽呈现在噬菌体表面 ,并建立了噬菌体表面呈现技术 (Phagedisplaytechniques)。在此基础上 ,1990年Scott首次将编码随机序列肽的DNA与丝状噬菌体外壳蛋白基因Ⅲ整合 ,使随机肽与噬菌体蛋白以融合形式表达 ,并呈现在噬菌体表面 ,从而建立了噬菌体随机肽库技术 (Phagedisplayrandom peptidelibraries)。近年来 ,噬菌体随机肽库已成为探索受体与配体之间相互作用结合位点、寻求高亲和力生物活性的配体分子、探测未知蛋白空间结构表位的…     

Introduction to antibody engineering and phage display

The vertebrate immune system, capable of rapidly producing highly specific antibodies upon immunisation, has been used to produce murine monoclonal antibodies (mAbs) via immortalisation and isolation of antibody-secreting cells. These mAbs have had a profound impact in the fields of diagnostics and therapeutics. However, the therapeutic use of murine mAbs is complicated by their immunogenicity. To circumvent this immunogenicity, antibody engineering techniques which render murine mAbs more compatible with the human immune system have been devised. Over the last decade, the technique of antibody phage display, which facilitates the production of human mAbs, has been developed. This review serves as an introduction to antibody engineering, phage display and the development of antibody fragments into viable diagnostic and therapeutic reagents.     

Protein-protein interactions in hematology and phage display

Phage display, which exploits fundamental tools and principles of immune repertoire diversity, antigen-antibody interactions, and clonal and immunologic selection, is used increasingly to advance experimental and clinical hematology. Phage display is based on the ability of bacteriophage to present engineered proteins on their surface coat. Diverse libraries of proteins such as peptides, antibody fragments, and protein domains corresponding to gene fragments or cDNAs may be displayed. Interactions between phage-displayed proteins and target antigens can be identified rapidly and characterized using high throughput methodologies. Peptide and gene fragment libraries are particularly useful to characterize binding interactions between proteins, such as ligand-receptor interactions. This approach allows rapid generation of human antibodies, often against nonimmunogenic, conserved proteins. Phage antibodies against surface and intracellular antigens are used as reagents for flow cytometry, in vivo imaging, and therapeutic targeting. Phage-derived antibodies also facilitate analyses of the humoral antibody response. Finally, cellular delivery of phage-displayed peptides and gene fragments can be used to modulate functional pathways and molecules in vitro and in vivo. The combinatorial power of phage display enables identification of candidate epitopes without knowledge of the protein interaction, a priori. Overall, these capabilities provide a versatile, high-throughput approach to develop tools and reagents useful for a plethora of experimental hematology applications. This paper focuses on current and future applications of antibody and epitope phage display technology in hematology.     

噬菌体展示技术的原理及应用

0 引言 1985年Smith首次证实丝状噬菌体fd基因组能通过基因工程的手段进行改造,将EcoR Ⅰ内切酶的部分基因片段(171 bp和132 bp)与pⅢ基因融合,获得的重组噬菌体可在体外稳定增生,表达产物能被抗EcoR Ⅰ内切酶抗体所识别。1988年Parmley et al将已知抗原决定族与p Ⅲ的N端融合呈现在表面,可特异性地被抗体选择出来,并提出通过构建随机肽库可以了解抗体识别的抗原决定簇表位的设想。1990年McCafferty et al地报道了用噬菌体展示技术筛选溶菌酶的单链抗体的方法,从而开始了这项技术的广泛应用的新时代。     

噬菌体随机十肽库的构建

目的采用丝状噬菌体表达载体,构建表达于丝状噬菌体尾丝蛋白P3N末端的随机十肽库。方法人工合成编码随机十肽的寡核苷酸片段及两条分别有SfiI和Notl酶切位点的引物,通过PCR反应得到其双链片段,将其克隆入载体pHEN1中,将重组产物转化入大肠杆菌TG1,构建成随机肽库。结果所建肽库含 1×10~9个集落形成单位(cfu)。随机挑取8个克隆测序,其核苷酸序列及推断出的氨基酸序列均随机。结论成功构建了有较高库容量的噬菌体随机十肽库。     

Preparation of single chain variable fragment of MG(7) mAb by phage display technology

AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.