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Bell, Matthew John. "Phage display of a cDNA library from Arabidopsis thaliana." Thesis, Coventry University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393652.
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Keklikian, Artine. "Construction of a Synthetic Human VL Phage Display Library and Isolation of Potential Neuropilin-1-specific VL Therapeutics from the Library." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/20197.
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Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration.
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Clayton, Ruth. "Generation and characterisation of human sperm antibodies by combinatorial phage display library technology." Thesis, University of Sheffield, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286988.
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Hald, Rikke. "Generation and characterisation of a naive human antibody phage display library : a resource for clinically relevant reagents /." Cph. : Department of Pharmacology, The Danish University of Pharmaceutical Sciences, 2004. http://www.dfh.dk/phd/defences/rikkehald.htm.
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Prendergast, D. "Discovery of tumour necrosis factor receptor-1 (p55) binding peptides using a phage display library." Thesis, Queen's University Belfast, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368468.
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Saxena, Abhishek. "Construction of immune scFv M13 phage display library and isolation of anti-glycan monoclonal antibodies." Thesis, University of Aberdeen, 2013. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=203295.
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Lim, Theam Soon [Verfasser]. "Parameters affecting phage display library design for improved generation of human antibodies / Theam Soon Lim." Berlin : Freie Universität Berlin, 2009. http://d-nb.info/1023748355/34.
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Cortini, Andrea. "Serum profiling and autoantibodies identification in Multiple Sclerosis using epitope and CSF IgG phage display libraries." Doctoral thesis, Università degli studi di Trieste, 2009. http://hdl.handle.net/10077/3073.
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2007/2008<br>Multiple sclerosis (MS) is considered the prototype of inflammatory autoimmune diseases of the central nervous system (CNS).
The typical feature of the disease is the plaques of demyelination. The evolution of the plaque lesion in MS implicates an inflammatory phase followed by a recovery of functional myelin; a second step is the chronic progressive disease with axonal loss. The earlier phase of MS may be mediated by an autoimmune reaction.
Whereas the role of T cells in MS pathogenesis is well established, the role of B cells and autoantibodies in demyelination and plaque formation is still unresolved. However several evidences suggest a contribute of autoantibodies in MS pathogenesis. B cells and myelin specific autoantibodies are present in the sclerosis plaques, and there is an increased production of immunoglobulin (Ig) in the cerebrospinal fluid (CSF) of more than 90% of MS patients . Typically these Ig present an oligoclonal pattern and sequencing of oligoclonal IgG showed extensive somatic mutations suggesting B cell clonal expansion and a specific antigen-driven immune response.
The most extensively studied putative autoantigens are components of CNS myelin (myelin basic protein MBP, proteolipid protein PLP, myelin oligodendrocyte glycoprotein MOG). The autoantibodies in MS recognize both linear and conformational epitopes, but at present the conformational epitopes of myelin proteins have not been identified. For example, in MS, the T-cell receptors of autoreactive T lymphocytes recognize various peptides of the MBP, and, in EAE, the anti-MOG antibodies recognize only conformational epitopes. Furthermore, the progression of MS is accompanied by the decline of primary T-cell autoreactivity and by the concurrent emergence of neo-autoreactivity (epitope spreading).
However recent investigation have showed that no myelin antigens, like neuron-specific enolase (NSE), retinal arrestin, beta-arrestin, may also have a role in MS pathogenesis. Autoimmunity against these antigens may be linked to neurodegeneration, defective remyelination, and predisposition to uveitis in multiple sclerosis.
Several strategies, involving the phage display technology, have been employed in the attempt to discover the antigen that drives the immune response in MS.
A first strategy depends on the cloning of IgG repertoire of MS patients in a phage display library screened with brain sections or known antigens. Another strategy involves large phage display libraries of random peptides screened with IgG of CSF in order to identify peptides recognized by antibodies present in CSF of MS patients.
Phage display is a technique which involves the coupling of phenotype to genotype in a selectable format. It has been extensively used in molecular biology to study protein-protein interactions and to select antibodies against a wide range of different antigens.
In this project we have proposed:
1. to study the autoimmune response in MS by using the phage display for the expression of antibodies involved in the disease. We wanted to make a ScFv library from B cells of CSF of different multiple sclerosis patients, to employ as tool to select a phage display Human Brain cDNA library for the identification of new antigens recognized by the immune sistem in MS patients.
2. To produce single gene mini library of putative antigens (MBP,PLP, MOG) for the generation of epitope chips to use for serotyping the immune response in different patients
3. To investigate the feasibility to use a single gene phage display mini-library as tool for epitope mapping (both linear and conformational) of novels autoantigens
4. To investigate the role of NSE(neuron specific enolase), a new possible no myelin autoantigen in multiple sclerosis, in the pathogenesis of the disease and the usefulness as possible diagnostic marker.
Results:
Scope 1
B-cells from liquor of two MS patients were centrifuged and the total RNA was extracted from the pellets. Total RNA was retrotranscripted and variable region of heavy and light chain of the antibodies were amplified by PCR. Heavy chain and light chain were assorted and assembled before to be cloned in the phagemid vector pDAN 5.
A 2x104 independent clones library was obtained and analyzed by PCR and fingerprinting. A diversity of 30,8% for heavy chain and 72,7% for light chain was established.
ScFv library was used to select a phage display Human Brain cDNA library.
17 clones with an high reactivity were obtained and after sequencing 6 clones on 17 have shown to be the same antigen(antigen A ); the reactivity on other two antigens obtained with the selection (antigen B and C) of CSF from 18 MS patients and 16 patients with other neurological disease (OND) was tested by ELISA to evaluate diagnostic value of this protein. The results shown that SM response was statistically different from OND response; the ELISA test gave a specificity of 94,12% and a significance of 53,85 %.
The reactivity for the antigen B was also evaluated on sera of MS patients and controls. The MS response was statistically different from OND response and shown a specificity of 97,44% and a significance of 58,62 %.
Scope 2&3
We have generated three single gene mini libraries of the major antigens in MS (MBP, MOG and PLP); cDNA of each gene was obtained by RT-PCR and after fragmentation cloned in a phagemid vector (pEP1) to obtain a mini-library for each gene.
We have obtained a 2x105 for MBP, 2.4x104 for MOG and 1.6x106 for PLP independent clones library. MBP and MOG libraries were characterized by PCR and fingerprinting. Sequencing analysis shown that the entire MBP transcript variant 7 mRNA (664-1177 nt) and MOG isoform alpha 1 mRNA (262-918 nt) were represented in the respective library.
To testing the capacity of selecting a single epitope from our libraries, we have performed a selection test with a commercial monoclonal antibody that recognize MBP 82-98 epitope; after three selection panning all selected clones contain the nucleotidic sequence 906- 956 nt (MBP transcript variant 7 mRNA) which encodes the immunogenic epitope recognized by the monoclonal antibody.
Scope 4
The reactivity of sera from 31 MS patients and 14 healthy controls was tested by ELISA on NSE ; statistical analysis of the results shown that the two populations were significantly different.<br>XXI Ciclo<br>1981
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Bosompem, Amma N. "Isolation of an anti-CD20 single chain variable fragment from a naïve human phage-scFv library." Connect to this title online, 2007. http://etd.lib.clemson.edu/documents/1202410076/.
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Marson, Lorena. "Phage-display epitope library development for biomarkers identification in autoimmune diseases of the Central Nervous System." Doctoral thesis, Università degli studi di Trieste, 2012. http://hdl.handle.net/10077/7405.
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2010/2011<br>The principal aim of my PhD was the setting of a protocol for the creation of phage libraries to display cDNA fragments encoding real ORF sequences, that could correspond to potential epitopes. A similar phage display library contains all the potential ORF repertoire of a cell or tissue. This tool can be specially used in the study of autoimmune diseases to perform different kind of analysis, such as the identification of epitopes involved in pathological reaction, the comparison between healthy and pathological conditions, or between different pathological conditions. A complex protocol was developed. It provides for: cDNA normalization, cDNA fragmentation to obtain peptides with useful size, and ORF enrichment to obtain really coding fragments. With this system we have created a epitopes library from Human brain mRNA.<br>Il principale obiettivo del mio lavoro di ricerca è la messa a punto di un protocollo per la costruzione di librerie fagiche di frammenti di cDNA codificanti per frammenti ORF, e che quindi potrebbero corrispondere a potenziali epitopi. Questo tipo di librerie contengono, potenzialmente, tutto il repertorio ORF di una cellula o di un tessuto e possono quindi essere utilizzate nello studio di malattie autoimmuni al fine di identificare nuovi epitopi coinvolti nella risposta immunitaria, di fare un confronto tra lo stato patologico e quello sano o tra diverse condizioni patologiche. Abbiamo quindi messo a punto un complesso protocollo che prevede: la normalizzazione del cDNA, la sua frammentazione per ottenere peptidi di dimensioni opportune, e l'arricchimento in frammenti realmente codificanti. Con questo sistema abbiamo realizzato una libreria di epitopi a partire da mRNA di cervello umano.<br>XXIV Ciclo<br>1984
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Abi, Ghanem Daad Ali. "Phage display selection of recombinant antibodies derived from a chicken immune library against cryopreserved Eimeria tenella sporozoites." [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1847.
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Sorsa-Leslie, Tarja. "Combining phage display antibody library and bioassay technologies to identify candidate gonadotropin surge attenuating factor (GnSAF) molecules." Thesis, University of Aberdeen, 2005. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU212837.
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The aim of this thesis was to generate "artificial" antibodies against a bioactive protein, GnSAF, produced by human ovarian granulosa cells that remains unidentified after 20 years of research. The library used in this study was a synthetic single-chain antibody scFv, Tomlinson J library. The antigen for biopanning was partially purified GnSAF. Screening the antibody clones from the library incorporated an additional selection step: an in vitro rat monolayer bioassay for GnSAF based on the specific suppression of GnRH-induced LH secretion. The initial screening with a binding ELISA technique resulted in 8 clones that were tested by bioassay, initially as pooled phage forms and subsequently as individual soluble scab forms. Three scabs recognised GnSAF bioactivity; with the suppression of GnRH-induced LH secretion by GnSAF-containing preparations reduced by up to 50% following incubation with the scabs. In order to improve the stability of the scabs for immunopurification purposes, and to widen the range of secondary labelled-antibodies available, the scabs were engineered into full length human immunoglobulins (IgG). One clone of the purified IgG form significantly altered GnSAF dose-response curves and demonstrated high affinity for GnSAF bioactivity when immobilised. When used for repeated immunopurification cycles and then Western blotted, this antibody enabled the isolation of a distinct band at around 66 kDa suggesting that this might be GnSAF. The main candidate molecules identified from the immunopurified material by excision of 2-D gel protein spots was a human serum albumin precursor and alloalbumin. This study demonstrates that the combination of bioassay and phage display technologies is a powerful tool in the study of uncharacterised proteins.
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Nguyen, Hai Phu. "Combination of hu-PBL-SCID mice and scFv phage display library, an effective alternative for hu-mAb production." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/NQ58617.pdf.
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Fehrsen, Jeanni. "Isolation of antigenic peptides of Cowdria ruminantium and their encoding genes using a genome-derived phage display library." Thesis, Rhodes University, 2003. http://hdl.handle.net/10962/d1003979.
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The development of new and effective vaccines and immunodiagnostic reagents requires the characterisation of antigenically relevant proteins and their interactions with the products of the immune system. Phage display technology was investigated as a means of elucidating some of the antigenic properties of the rickettsial parasite, Cowdria ruminantium (Cowdria). Randomly fragmented gene-derived libraries have been useful in elucidating viral and other epitopes, but only limited work has been done with entire genomes. A phage display library expressing a repertoire of Cowdria peptides was constructed. It was sufficiently large to represent the organism's genome, but lacked phages displaying peptides coded for by genes containing a Pvu II restriction enzyme site, including the one coding for the major antigenic protein 1 (MAP1). This was considered advantageous since MAP1 is immunodominant and has already been well characterised. Affinity selection with antibodies against Cowdria proteins other than MAP1 allowed several antibody-reactive peptides to be isolated. These selected sequences were placed in the context of the genome by screening a lambda bacteriophage library and by comparison with Cowdria DNA sequences. Apart from showing that antigenic mimics were present in the phage display library, six open reading frames encoding putative Cowdria proteins were identified. All had similarities to, or motifs in common with, membrane proteins and are thus likely to be exposed to the host's humoral immune system. Some of the proteins identified were larger than the antigens used to elicit the antibodies used for selection, probably as a result of the presence of cross-reactive epitopes. Despite limitations experienced when extending a fragmented-gene approach for epitope location to genomes, it was possible to identify an antigenic region on MAP1 by comparison with selected mimics. In addition, binding peptide sequences were identified with two monoclonal antibodies that had been raised against non-Cowdria antigens. An epitope on the VP7 protein of bluetongue virus was identified and peptides were found that reacted with a monoclonal antibody directed against malignant catarrhal fever virus. Thus, apart from being able to identify several potentially important Cowdria epitopes and genes, the fragmented-genome library holds promise as a universal reagent for identifying useful mimics.
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Bessa, Juliana Mattos de Almeida. ""Determinação de alvos antigênicos na doença reumática cardíaca utilizando phage display"." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/5/5146/tde-12042006-164408/.
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Pacientes com doença reumática cardíaca (DRC) desenvolvem lesões valvares mediadas por linfócitos T CD4+, capazes de reconhecer cruzadamente proteínas cardíacas e estreptocócicas pelo mecanismo de mimetismo molecular. Neste trabalho empregamos uma biblioteca peptídica de Phage Display para identificar auto-antígenos cardíacos capazes de serem reconhecidos por duas linhagens intralesionais de linfócitos T e um clone derivado de uma das linhagens isolados de pacientes com DRC. A análise dos peptídeos dos fagos em banco de dados de proteínas revelou novos epitopos da miosina cardíaca, laminina, vimentina e outras proteínas coiled-coil, provavelmente involvidos no processo auto-imune da DRC. Outras moléculas inflamatórias como citocinas, integrinas e fatores de crescimento também foram identificadas<br>Rheumatic heart disease (RHD) patients develop valvar lesions with CD4+ T lymphocytes infiltrating the heart. Molecular mimicry between streptococcal and cardiac proteins recognized by these T cells may explain these auto-aggressive lesions. In the present work we used a Phage Display peptide library to identify cardiac antigens which could be recognized by two heart infiltrate T cell lines and by a T cell clone derived from one of the lines which were isolated from RHD patients. Using the protein data bank to analyse the phage peptides, we observed that many sequences showed homology with cardiac myosin, laminin, vimentin and other coiled-coil proteins, suggesting the involvement of these proteins in the autoimmune process of RHD. Other inflammatory molecules such as cytokines and integrins were also identified
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Igloi, Zsofia. "Identifying novel interaction partners for the hepatitis C virus NS5A protein by screening a human SH3 domain phage display library." Thesis, University of Leeds, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.574514.
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The hepatitis C virus is a worldwide cause of liver disease by infecting approximately 2 % of the population. One of its non-structural protein, NS5A, Is known to interact with many cellular proteins involved in cell signaling pathways. Doing so modifies the outcome of the signalling for the benefit of virus replication and survival. We have previously demonstrated that NSSA contains two Class II poly-proline motifs in the second low complexity sequence, with the consensus sequence Pro-X-X-Pro-X-Lys/ Arg, which mediate the interaction with SH3 domain containing proteins. Substitution of the prolines with alanines did not have an effect on RNA replication but disrupted interactions with SH3 domain containing proteins. In order to identify novel NSSA binding partners, genotype 2a (JFH1) NSSA was expressed with a biotin tag individually or in the context of the replicon as an experimental requirement to screen an all human SH3 domain displaying library. In total, sixteen SH3 domain containing binding partners were identified, from which Mlk3, RelA, Vinexin, PACSIN1 RBP2 and STAC1 were novel targets. The interaction of NS5A with Amph1, CMS, Nck1 and Ponsin were investigated in more details. Interactions of NS5A with Amph1 and CMS were confirmed using immunoprecipitation, immunofluorescence and biochemical assays. Interestingly these proteins have been implicated in clathrin-mediated endocytosis and epidermal growth factor receptor trafficking. Upon overexpression of wt but not the proline mutant NSSA the cellular localization of Amph1 was altered and interaction with dynamin2, a well characterized partner of Amph1, was disrupted. Furthermore, as it was reported before, NS5A diverts the epidermal growth factor receptor away from the late endosomes thereby modifying its degradation rate and signalling capability towards amongst other targets the Ras-Erk mitogen-activated protein kinase pathway, a signalling cascade known to be inhibited by NS5A in a PxxPxR dependent manner. NS5A-CMS and EGFR was found to co-localise to vesicles upon EGF stimulation and overexpression of CMS was found to reverse the effect of NS5A on EGFR.
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Caucheteur, Déborah. "Nouveau format de banques d’anticorps recombinants humains pour un criblage fonctionnel à grande échelle." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT012.
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Les anticorps monoclonaux (mAb) sont depuis les années 2000 devenus des médicaments incontournables et de routine en thérapie et notamment en cancérologie. Le domaine continue à croître très rapidement et devant l’abondance des molécules disponibles, il est de plus en plus important d’apporter des molécules innovantes à haute valeur ajoutée pour la thérapie. Deux grandes approches sont utilisées pour sélectionner ces mAbs : l’hybridation lymphocytaire à partir de souris normales ou humanisées ; Les systèmes de display comme le phage-display. Les intérêts majeurs du phage display sont la rapidité de développement des mAbs, la facilité de manipulation chez E. coli, l’accès aux techniques de "protein engineering". Classiquement, les anticorps sont d’abord sélectionnés sur leur capacité de liaison à l’antigène puis ensuite testés pour leur efficacité fonctionnelle dans des modèles cellulaires. Cependant, seulement une partie de l'activité des anticorps est expliquée par leur liaison à l’antigène et l’activité thérapeutique dépend aussi fortement de leur capacité à recruter le système immunitaire (ADCC) et à activer la cascade du complément (CDC).Ce projet de thèse consiste à développer un nouveau format de banque d'anticorps recombinants combinant la puissance de la sélection par phage display à un criblage fonctionnel au format IgG entières produites en cellules eucaryotes. Ce nouveau système est basé sur des régions initiatrices hybrides contenant à la fois des promoteurs et des séquences signal procaryotes et eucaryotes permettant l’expression dans ces deux systèmes cellulaires, et des évènements de recombinaisons sites-spécifiques transférant le fragment Fab du vecteur de display vers le chromosome d’une lignée cellulaire de mammifère spécialement développée pour aboutir à la sécrétion d’un anticorps humain monoclonal par la cellule. L’approche habituelle de reclonage un par un du vecteur E. Coli au format IgG n’est plus nécessaire puisqu’il se fait directement par transfection. Ce nouveau système rend possible le couplage d’une sélection par phage display à un criblage fonctionnel direct sur une large population de clones monoclonaux humains<br>Since 2000, monoclonal antibodies (mAb) have become essential and routine drugs in therapy and particularly in oncology. The field continues to grow very quickly and given the abundance of molecules available, it is increasingly important to bring innovative molecules with a high added value for therapy.Two main approaches are used to select these mAbs: hybridoma technology using normal or humanized mice; display systems such as phage-display. The major interests of phage display are the speed of mAb development, the facilities offered by E. coli and the easy access to protein engineering techniques. Typically, antibodies are first selected on their ability to bind to the antigen, and then tested for their functional efficiency in cellular models. However, only a part of the activity of antibodies is explained by their binding to the antigen, and the therapeutic activity also depends strongly on their ability to recruit the immune system (ADCC) and activate the complement cascade (CDC).This thesis project consists in the development of a new recombinant antibody library format combining the power of phage display selection with functional screening in a whole IgG format produced in eukaryotic cells. This new system is based on hybrid promoter and signal peptide regions allowing expression both in prokaryotic and eukaryotic cells, and a site-specific recombination event that exchanges the Fab between the display vector and the chromosome of an especially developed mammalian cell line resulting in the secretion of a monoclonal human antibody by the cell. The usual approach of recloning one by one from E.Coli vector to an IgG format is no more needed since it is done directly by transfection. This new system makes possible to couple selection by phage display with a direct functional screening of a large population of human monoclonal clones
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Ndlovu, Siphumelele. "The isolation of single chain variable region fragments (scFvs) from a phage display library, and expression of the isolated scFvs in Nicotiana benthamiana." Master's thesis, Faculty of Science, 2020. http://hdl.handle.net/11427/32303.
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Monoclonal antibodies (mAbs) are an important tool for both therapeutic and nontherapeutic applications. Their increased demand is due to their ability to recognize and bind specifically to a wide range of antigens. In addition to full-size antibodies, one can also utilise smaller antibody fragments, single chain variable region fragments (scFvs), which like full-size mAbs, are also capable of specific antigen-binding. The constant and rapidly expanding use of antibodies and their derivatives presents a need for a fast and effective method of production. Traditionally, antibodies have been produced using hybridoma technology. They have also been successfully produced in other expression hosts such as bacteria, yeasts, insect cells and mammalian cell lines. However, these expression systems come with a few disadvantages, some of which include high maintenance costs as well as lengthy and laborious production protocols. This dissertation describes the use of phage display technology to screen for and identify scFvs that bind to three different test antigens. Phage display library technology involving the expression and presentation of antibody or antibody derivatives on the coat surfaces of phage particles. It is considered to be a preferable alternative to hybridoma technology because it eliminates the requirement for immunization of animals, making it a more rapid and animal-friendly method for the production of antibodies compared to that of hybridoma technology. A naïve mouse scFv phage display library was screened with appropriate antigens to isolate scFvs which bind to rabbit IgG, human IgG and the Shuni virus (SHUV) N protein. Isolated scFvs were sequenced, cloned and tested for binding to their cognate antigens using phage ELISA, phage dot blots and phage western blots. ScFvs displaying the highest affinities for their respective antigens were selected for cloning and expression in plants, as this expression system is scalable, cheaper, safe and facilitates posttranslational modifications to recombinant proteins such as glycosylation. Rabbit IgG and human IgG scFvs were isolated successfully from the mouse scFv phage library, however, successful binding of the scFvs to the respective antigens by western blotting and ELISAs was not demonstrated. On further investigation, it appeared that the protocols were flawed, as the secondary anti-mouse AP conjugate, iv used in the western blots and ELISAs was found to cross-react with both rabbit and human IgG. Since we were not able to pinpoint scFvs with high binding affinity, the mouse phage display library was screened for scFvs that bound to SHUV N protein instead. This was more successful in that several scFvs with high binding affinity were isolated. Three scFvs with the highest binding affinity for the SHUV N protein were selected and their nucleotide sequences determined. Due to time constraints only 2 of the identified scFvs were selected for further cloning and expression in plants. Both scFvs were cloned into the pTRA-HRPB2SEKDEL plant expression vector that contains the gene sequence for a his6x tag to assist with downstream purification as well as a horse radish peroxidase (HRP) gene. Cloning scFvs into this vector allows their fusion to HRP, resulting in the production of potential reagents for use as secondary antibodies in western blots and ELISAs. The cloned scFvs were expressed transiently in tobacco plants using Agrobacterium-mediated infiltration. Plant expression of the HRP-fused scFvs was optimized; both were optimally expressed at 5 days post infiltration (dpi) when co-expressed with a silencing suppressor (pBIN-NSs). Extraction of the scFvs from the plants was most effective when a bicine buffer with a pH of 8.4 was used. Partial purification of the scFvs was achieved by isoelectric and ammonium sulphate precipitation. Preliminary tests were done to test functionality of the partially purified scFvs, in which the ability of the scFvs to recognize and bind to the SHUV N protein in a dot blot was tested. However, both were found to be non-functional in this regard. Further investigation into the reason for the demonstration of non-functionality showed that the HRP was being spontaneously cleaved from the scFv. This study demonstrates that it is possible to isolate antigen-specific scFvs from a phage display library. However, their binding capacity needs to be analysed fully prior to incorporating them into fusion proteins which can be used as potential diagnostic reagents.
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Bandmann, Nina. "Rational and combinatorial genetic engineering approaches for improved recombinant protein production and purification." Doctoral thesis, Stockholm : Bioteknologi, Kungliga Tekniska högskolan, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4318.
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Hubert, Ágnes [Verfasser], Wolfgang [Akademischer Betreuer] Baumeister, and Sevil [Akademischer Betreuer] Weinkauf. "Needle in the haystack: Protein complex purification from Thermoplasma acidophilum with a phage display library / Ágnes Hubert. Gutachter: Sevil Weinkauf ; Wolfgang Baumeister. Betreuer: Wolfgang Baumeister." München : Universitätsbibliothek der TU München, 2013. http://d-nb.info/103576671X/34.
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Mundin, Georgia Sabio Porto. "Identificação de marcadores moleculares para células T reguladoras humanas com perfil CD4+CD25+ por phage display." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/5/5146/tde-28012009-131608/.
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Há dados na literatura indicando que as células que saem do timo com o fenótipo CD4+CD25+ são desenvolvidas continuamente como uma linhagem independente e possuem um papel importante no processo de regulação da resposta imune. Essas células são chamadas células T reguladoras naturais. Várias questões sobre estas células permanecem em aberto, como por exemplo, como elas são geradas, o que é determinante na sua atividade reguladora e que marcadores específicos podem ser usados para identificá-las? Dentro deste contexto, o nosso objetivo neste trabalho foi identificar no timo e em timócitos CD4+/CD25+ humanos, novas moléculas potencialmente importantes no desenvolvimento e/ou na atividade supressora das células T reguladoras naturais. Para este objetivo, utilizamos a abordagem de phage display, com uma biblioteca de fagos de peptídeos, e timos humanos obtidos de pacientes portadores de cardiopatias congênitas, submetidos a cirurgias cardíacas realizadas no InCor. A busca dessas moléculas foi feita, separadamente, em 3 tipos de material biológico: timócitos totais, fragmento do tecido tímico e timócitos CD4+/CD25+. Antes da incubação da biblioteca de fagos com os timócitos totais e timócitos CD4+/CD25+ (separação em FACS), foi realizada uma etapa de preclearing, incubando-se a biblioteca de fagos com um pool de células mononucleares de sangue periférico (PBMC) ou timócitos CD4+/CD25-, respectivamente. Os fagos não ligantes, recuperados desta etapa, foram então incubados com as células de interesse. Para o tecido tímico não foi feita etapa de pre-clearing. Os fagos obtidos com os diferentes materiais biológicos foram recuperados em cultura de bactérias e usados em ciclos posteriores de seleção. Após três ciclos de seleção, os fagos foram seqüenciados e identificados quanto à expressão de peptídeos ligantes para timócitos totais, timo e timócitos CD4+/CD25+, e analisados em bancos de dados no BLAST. Os fagos selecionados para validação um ligante de tecido tímico: M2C e um ligante de timócitos CD4+/CD25+: R2A fazem similaridade a duas proteínas associadas ao metabolismo da Vitamina D3, molécula envolvida em imunorregulação e indução de tolerância, em diversos modelos experimentais. Porém, não há dados na literatura a respeito do seu papel em células T reg naturais. Na validação molecular desses fagos, apesar de certa variabilidade entre os diferentes ensaios, verificamos, por ELISA, que os fagos se ligam preferencialmente a 1,25 diidroxivitamina D3, forma ativa da Vitamina D3. Entretanto, nos ensaios de validação funcional, a influência da vitamina D na diferenciação dessas células não foi confirmada de forma consistente, uma vez que só tivemos aumento no número de células CD4+/CD25+, em cultura com Vitamina D, em poucos experimentos. As moléculas identificadas no presente estudo podem ter implicações relevantes no processo de diferenciação e na atividade de células T CD4+CD25+ reguladoras e serão mais investigadas na continuidade deste trabalho.<br>There are consistent data in literature indicating that thymic CD4+CD25+ cells play an important role in immune regulation and are continuously developed as an independent lineage in the thymus. These cells are known as natural regulatory T cells. Several questions about these cells remain unanswered, such as how they are generated, what is determinant in their regulatory function and which specific molecular markers can be used to identify them. Taking this into consideration, our aim was to identify new potentially important molecules in the development and/or supressive function of natural regulatory T cells, both in the thymus and in CD4+CD25+ thymocytes. For this, the phage display technique was employed, with a peptide phage library and thymic specimens obtained from children who underwent corrective cardiac surgery at the Heart Institute (InCor), in São Paulo. The search for these molecules was separately performed in 3 types of biological material: thymic tissue, thymocytes and CD4+CD25+ thymic cells. In the first stage, the phage peptide-library was incubated with a pool of PBMC (peripheral blood mononuclear cells). After the incubation, phages bound to PBMC were discarded (pre-clearing). In the second stage, unbound phages were incubated with either total thymocytes or CD4+CD25+ thymic cells. The pre-clearing stage was not perfomed in the thymic tissue. The phages obtained with after incubation with the different biological materials were recovered in E. coli culture and used in additional cycles of selection. After three rounds of selection, the recovered phages from the total thymocytes, from thymic tissue and thymocytes CD4+CD25+ were sequenced and their ligands identified. Among the phages selected for validation one ligand of thymic tissue: M2C and one ligand of CD4+CD25+ thymocytes: R2A present similarity to two proteins associated to the metabolism of Vitamin D3, a molecule involved in imunoregulation and toelrance induction in several experimental models. However, there are no data in the literature concerning the possible role of this moelcule in natural regulatory T cells. In the molecular validation of theses phages, although some variability between the diffeterent assays we have verified by ELISA, that the phages present preferential binding to the 1,25 dhydroxyvitamin D3, the active form of Vitamin D3. However, in the functional validation assays, the influence of the Vitamin D3 in the differentiation of these cells could not be consistently confirmed since we could observe an increase in the number of CD4+CD25+ cells cultured with vitamin D in only a few experiments. The ligand-receptor molecules we have defined in this study may have relevant implications in the development of CD4+CD25+ regulatory T cells in the thymus
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Sulaiman, Lanre Precieux Kabir. "Seleção de motivos semelhantes a Papilomavírus, a partir de bibliotecas de phage display, que apresentem potencial aplicação translacional." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-29012018-133017/.
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O vínculo entre papilomavírus humano de alto risco e câncer cervical está bem estabelecido. Apesar da existência de vacinas profiláticas contra infecções pelos tipos mais comuns de HPV, para infecções e tumores causados por esses vírus as alternativas terapêuticas são restritas. Encontramos alguns motivos com homologias para proteínas do HPV de alto risco durante o imunoscreening de uma biblioteca de phage display com soros de participantes HPV-16-soropositivos da coorte Ludwig-McGill. Após enriquecimento das sequências, os bacteriófagos recombinantes foram purificados e amplificados para uso como imunógenos.Usando uma abordagem profilática, nós vacinamos experimentalmente camundongos imunocompetentes com um dos nossos bacteriófagos recombinantes, usando o bacteriófago sem inserto como controle. Estes camundongos foram então desafiados com células tumorais TC-1 (HPV-16 positivas), tendo-se avaliado as respostas imunes disparadas durante a progressão tumoral. Também usamos uma abordagem terapêutica, aonde os camundongos foram primeiro injetados com as células tumorais e imunizados com o bacteriófago após o estabelecimento do tumor. O crescimento tumoral foi monitorado e os tumores, baço e linfonodos foram avaliados quanto à quantidade e qualidade da resposta imunológica. Os testes de ELISA revelaram que todos os camundongos vacinados responderam à imunização com os diferentes bacteriófagos. O crescimento tumoral foi significativamente reduzido nas imunizações profiláticas e terapêuticas, embora a redução do tumor fosse mínima quando os camundongos foram tratados 9 dias após o enxerto. A redução no crescimento tumoral também se traduziu em uma sobrevivência significativamente maior para os camundongos imunizados. Estudos de infiltração celular não revelaram alterações em diversas sub-populações imunes, mas uma tendência de aumento de linfócitos T citotóxicos foi observada nos camundongos imunizados com PEP1 (bacteriófago contendo inserto). A importância deste aumento de CD8 na redução observada do crescimento tumoral foi confirmada utilizando camundongos CD8-knockout, onde a redução do crescimento tumoral previamente observada foi anulada. Foi observado um aumento de taxa CD8:CD4 nos camundongos imunizados e isto é uma indicação de ambiente tumoral citotóxico. Os ensaios de proliferação celular para testar a especificidade do antígeno dos linfócitos dos camundongos imunizados foram, no entanto, inconclusivos; da mesma forma, não pudemos alterar o padrão observado com o uso de adjuvante CpG. A utilidade da técnica de phage display também foi observada neste trabalho experimental. Trabalhos adicionais para entender o mecanismo de ação desses fagos recombinantes no controle do crescimento de tumores causados por HPV e seu potencial imuno-estimulador são necessários<br>The link between high-risk human papillomavirus and cervical cancer is well established. Despite the existence of prophylactic vaccines against infections by the most common types of HPV, therapeutic alternatives are limited for infections and tumors caused by these viruses. We found some homology motifs for high-risk HPV proteins during the immune-panning of a phage display library with sera from HPV-16- seropositive participants of the Ludwig-McGill cohort. After enrichment of the sequences, the recombinant bacteriophages were purified and amplified for use as immunogens. Using a prophylactic approach, we vaccinated experimentally immunocompetent mice with one of our recombinant bacteriophages using the insertless bacteriophage as a control. These mice were then challenged with TC-1 tumor cells (HPV-16 positive), and the immune responses triggered during tumor progression were evaluated. We also used a therapeutic approach where mice were first injected with tumor cells and immunized with the bacteriophage after tumor establishment. Tumor growth was monitored and tumors, spleen and lymph nodes were evaluated for the quantity and quality of the immune response. ELISA tests revealed that all vaccinated mice responded to immunization with the different bacteriophages. Tumor growth was significantly reduced in prophylactic and therapeutic immunizations, although tumor reduction was minimal when mice were treated 9 days after TC-1 cells grafting. The reduction in tumor growth also translated into a significantly greater survival for the immunized mice. Cell infiltration studies did not reveal changes in several immune subpopulations, but an upward trend in cytotoxic T lymphocytes was observed in mice immunized with PEP1 (insert-containing bacteriophage). The importance of this increase in CD8 in the observed reduction of tumor growth was confirmed using CD8-knockout mice, where the previously observed reduction of tumor growth was abolished. An increase in CD8:CD4 rate was observed in the immunized mice and this is an indication of a cytotoxic tumor environment. Cell proliferation assays to test the antigen specificity of lymphocytes from immunized mice were, however, inconclusive; likewise, we could not change the pattern observed with the use of CpG adjuvant. The usefulness of the phage display technique was also observed in this experimental work. Additional studies to understand the mechanism of action of these recombinant phages in the control of HPV tumor growth and its immunostimulatory potential are warranted
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Zou, Jun. "Characterization of peptides and phage that bind galectin-3 selected from bacteriophage display libraries a study of the role of galectin-3 in metastasis-associated cancer cell adhesion /." Diss., Columbia, Mo. : University of Missouri-Columbia, 2005. http://hdl.handle.net/10355/4149.
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Thesis (Ph. D.)--University of Missouri-Columbia, 2005.<br>"December 2005" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.
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Bernedo-Navarro, Robert Alvin 1975. "Obtenção de peptídeos com capacidade inibitória da ação citotoxigênica das toxinas Stx de Escherichia colia partir de bibliotecas de phage display." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314705.
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Orientador: Tomomasa Yano<br>Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia<br>Made available in DSpace on 2018-08-23T11:52:53Z (GMT). No. of bitstreams: 1
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Previous issue date: 2013<br>Resumo: Escherichia coli produtora de toxina de Shiga (STEC) é um grupo de importantes patógenos para humanos. Essas bactérias são relacionadas a várias doenças, como por exemplo, Síndrome Urêmica Hemolítica e produzem potentes toxinas denominadas toxinas de Shiga. Essas toxinas, tanto Stx1 quanto Stx2, compartilham um receptor celular comum, a globotriaosilceramida (Gb3) e exibem a mesma atividade biológica intracelular. O desenvolvimento de novos agentes neutralizantes dos danos induzidos por Stx pode representar uma estratégia promissora para o tratamento das doenças causadas por STEC em humanos. No presente estudo, nós desenvolvemos peptídeos sintéticos que exibem atividade neutralizante contra a citotoxicidade induzida por Stx tanto in vitro quanto in vivo e, além disso, que se ligam eficientemente ao receptor Gb3. O peptídeo P12-26 compete eficientemente com Stx2 para a ligação ao Gb3 in vitro. Além disso, os peptídeos PC7-12, P12-26 e PC7-30 inibiram a citotoxicidade de Stx1 e Stx2 em células Vero. Nós observamos que o peptídeo PC7-30 em forma de loop e o peptídeo P12-26 que é linear produziram as maiores porcentagens de inibição de Stx1 e Stx2 em células Vero, respectivamente. No entanto, o peptídeo P12-26 não inibiu a letalidade em camundongos, enquanto que o peptídeo PC7-30 inibiu a letalidade causada pela toxina Stx1. Nossos resultados indicam que os peptídeos P12-26 e PC7-30 são candidatos promissores para o desenvolvimento de agentes terapêuticos contra as doenças em seres humanos causadas por STEC<br>Abstract: Shiga toxin-producing Escherichia coli strains are important pathogens for humans. These bacteria are linked with severe diseases such as hemolytic uremic syndrome and produce potent known as Shiga toxins. These toxins, Stx1 and Stx2, share a common cellular receptor called globotriaosylceramide (Gb3) and exhibit the same intracellular biological activity. The development of new neutralizing agents for Stx-induced damage may represent a promising strategy for the treatment of diseases caused by STEC infections. In this study, we developed synthetic peptides that exhibit neutralizing activity against Stxinduced cytotoxicity both in vitro and in vivo and that bind efficiently to the Gb3 receptor. The peptide P12-26 competed efficiently with Stx2 for binding to Gb3 in vitro. Moreover, the peptides PC7-12, P12-26 and PC7-30 inhibited the cytotoxicity of Stx1 and Stx2 in Vero cells. We observed that the loop-constrained peptide PC7-30 and linear peptide P12-26 produced higher percentages of inhibition of Stx1 and Stx2 in Vero cells, respectively. However, the peptide P12-26 did not inhibit lethality in mice, whereas the loopconstrained peptide PC7-30 inhibited the lethality caused by Stx1. Our results indicate that the peptides P12-26 and PC7-30 are promising candidates for the development of therapeutic agents against diseases caused by STEC in humans<br>Doutorado<br>Bioquimica<br>Doutor em Biologia Funcional e Molecular
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Lehtiö, Janne. "Functional studies and engineering of family 1 carbohydrate-binding modules." Doctoral thesis, KTH, Biotechnology, 2001. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3211.
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<p>The family 1 cellulose-binding modules (CBM1) form a groupof small, stable carbohydrate-binding proteins. These modulesare essential for fungal cellulosedegradation. This thesisdescribes both functional studies of the CBM1s as well asprotein engineering of the modules for several objectives.</p><p>The characteristics and specificity of CBM1s from the<i>Trichoderma reesei</i>Cel7A and Cel6A, along with severalother wild type and mutated CBMs, were studied using bindingexperiments and transmission electron microscopy (TEM). Datafrom the binding studies confirmed that the presence of onetryptophan residue on the CBM1 binding face enhances itsbinding to crystalline cellulose. The two<i>T. reesei</i>CBM1s as well as the CBM3 from the<i>Clostridium thermocellum</i>CipA were investigated by TEMexperiments. All three CBMs were found to bind in lineararrangements along the sides of the fibrils. Further analysesof the bound CBMs indicated that the CBMs bind to the exposedhydrophobic surfaces, the so called (200) crystalline face of<i>Valonia</i>cellulose crystals.</p><p>The function and specificity of CBM1s as a part of an intactenzyme were studied by replacing the CBM from the exo-actingCel7A by the CBM1 from the endoglucanase Cel7B. Apart fromslightly improved affinity of the hybrid enzyme, the moduleexchange did not significantly influence the function of theCel7A. This indicates that the two CBM1s are analogous in theirbinding properties and function during cellulosehydrolysis.</p><p>The CBM1 was also used for immobilization studies. Toimprove heterologous expression of a CBM1-lipase fusionprotein, a linker stability study was carried out in<i>Pichia pastoris</i>. A proline/threonine rich linker peptidewas found to be stable for protein production in this host. Forwhole bacterial cell immobilization, the<i>T. reesei</i>Cel6A CBM1 was expressed on the surface of thegram-positive bacteria,<i>Staphylococcus carnosus</i>. The engineered<i>S. carnosus</i>cells were shown to bind cellulosefibers.</p><p>To exploit the stable CBM1 fold as a starting point forgenerating novel binders, a phage display library wasconstructed. Binding proteins against an amylase as well asagainst a metal ion were selected from the library. Theamylase-binding proteins were found to bind and inhibit thetarget enzyme. The metal binding proteins selected from thelibrary were cloned on the surface of the<i>S. carnosus</i>and clearly enhanced the metal bindingability of the engineered bacteria.</p><p><b>Keywords</b>: cellulose-binding, family 1carbohydrate-binding module, phage display, bacterial surfacedisplay, combinatorial protein library, metal binding, proteinengineering,<i>Trichoderma reesei, Staphyloccus carnosus</i>.</p>
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Grimm, Sebastian. "Ribosome display for selection and evolution of affibody molecules." Doctoral thesis, KTH, Molekylär Bioteknologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-33191.
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Affinity proteins are invaluable tools in biotechnological and medical applications. This thesis is about combinatorial protein engineering principles for the generation of novel affinity proteins to purify mouse immunoglobulin, detect a potential cancer marker protein or inhibit a cell proliferation pathway. In a first study, ribosome display was for the first time applied to the selection of so-called affibody molecules, including the design of a ribosome display gene cassette, initial test enrichment experiments and the selection of binders against murine IgG1. One of the selected binders (ZMAB25) showed a highly selective binding profile to murine IgG1, which was exploited in the recovery of two different mouse monoclonal IgG1 antibodies from a bovine immunoglobulin-containing background. Ribosome display was further applied to the selection of affibody molecules binding to SATB1, a suggested marker protein for metastasizing adenocarcinoma. The study also included the selection of VHH antibody fragments from a naïve gene repertoire displayed on phage. Binders from both classes of protein scaffolds could be isolated that selectively recognized SATB1 but not its close homologue SATB2, and were used to detect endogenous SATB1 in Jurkat cells by immunofluorescence microscopy. The well-established phage display technology was used to select affibody molecules binding to H-Ras and Raf-1, both involved in the mitogen-activated protein kinase (MAPK) pathway and playing a central role in the control of cell proliferation, survival and differentiation. An isolated affibody molecule denoted ZRAF322 was found to selectively bind to Raf-1 and inhibit the interaction between H-Ras and Raf-1 in vitro. In a continued effort, ribosome display was applied to the affinity maturation of the ZRAF322 variant in a novel approach, based on repetitive cycles of diversification by error-prone PCR of the entire affibody gene and ribosome display selection, mimicking the principles of natural evolution. The method involved a monitoring of the progress of evolution and variants of ZRAF322 with 13- to 26-fold improved affinities were obtained, that contained different combinations of single or double amino acid substitutions in either previously randomized or framework positions. Implications of the substitutions for binder stability and selectivity were also investigated, showing that a higher affinity could be associated with a lower thermal melting point and that affinity-improved variants showed uncompromised binding selectivity to the hRaf-1 target.<br>QC 20110506
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Shahsavarian, Melody. "Genesis of immune diversity and selection of catalytic antibodies : a new investigation." Thesis, Compiègne, 2015. http://www.theses.fr/2015COMP2215/document.
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Les anticorps catalytiques (ou abzyme) ont fait l’objet de nombreuses recherches et ont été produits pour réaliser de nombreuses réactions. Ces protéines ont été ensuite découvertes dans le sérum d’individus sains ou atteints de pathologies, dont les pathologies autoimmunes. Les études suggèrent que ces abzymes peuvent avoir des effets bénéfiques ou délétères sur la santé des individus. L’origine des anticorps catalytiques et leur rôle restent ambigus et doivent être approfondis. Nous avons développé une nouvelle stratégie visant à étudier les abzymes, basée sur la technologie du phage display. Nous avons construit 4 banques de fragments d’anticorps, chacune présentant un répertoire immun différent (fond génétique et état d’immunitaire) : saine et naïve, saine et immunisée, autoimmune et naïve, et autoimmune et immunisée. Les stratégies d’amplification et de clonage des régions variables des immunoglobulines ont été conçues afin d’optimiser la taille et la diversité des banques. Nous avons rassemblé les 4 banques en une banque unique élargie contenant 2.7×109 séquences. L’analyse des séquences a mis en évidence des différences dans les profils d’expression des sous-groupes de gènes selon la banque. Nous avons ensuite procédé à la sélection d’abzymes à activité β-lactamase en utilisant deux cibles : un peptide cyclique, et un dérivé de sulfone pénam, inhibiteurs de l’enzyme. Nous avons sélectionné 5 abzymes. Chacun de ces immunoglobulines ont des séquences protéiques propres, incluant un potentiel site actif. Ces résultats montrent que différents motifs peuvent assurer la fonction catalytique de la β-lactamase, confirmant la flexibilité moléculaire de cette enzyme<br>Catalytic antibodies (or abzymes) have been the focus numerous studies for some decades and have been produced with the ability to catalyze a wide range of reactions. They have also been discovered naturally in normal physiological and pathological conditions, notably on the background of autoimmune disease. Some have beneficial effects and others are detrimental to individual’s health. Hence, the origin of abzymes and their role in the immune response are ambiguous and must be enhanced. We have developed a novel strategy for the study of abzymes based on the phage display technology. We have constructed 4 libraries representing 4 murine immune repertoires with different genetic backgrounds and immunological states : healthy and naïve, healthy and immunized, autoimmune and naïve , and autoimmune and immunized. The strategies for the amplification and cloning of the immunoglobulin (lg) variable regions have been designed to optimize the size and diversity of the libraries. We have been able to pool the four libraries to create a large repertoire of size 2.7x109. After sequence analysis, we have found a number of statistically significant differences between the libraries. We have then used two strategically chosen targets to select for antibodies endowed with β lactamase activity : a cycle peptide and a penam sulfone, both inhibitors of the enzyme. We have selected for a total of 5 lgs endowed with β lactamase activity. The selected abzymes have different amino acid sequences. 3D modeling has provides insights on potential active sites demonstrating the ability of different structures to maintain the β lactamase activity and confirming the flexibility of the active site
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Harris, Jasmine K. "Bioconjugation of aminated DNA as a method of rapid polymer library generation for Corona Phase Molecular Recognition." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/119063.
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Thesis: S.B., Massachusetts Institute of Technology, Department of Materials Science and Engineering, 2018.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (page 39).<br>An experimental study was performed to determine the effects on Corona Phase Molecular Recognition (CoMoRe) of bioconjugating a host of small molecules to DNA wrapped single-walled carbon nanotubes. In addition, the study observed the effects of the DNA sequence length on the subsequent effectiveness of the small molecules to alter the corona phase. The conjugation of small molecules was shown to alter both the intensity and the position of the fluorescence and absorbance profile. The length of the DNA sequence was found to change the small molecule's ability to alter the fluorecence spectra of the wrapped nanotubes. The EDC/sulfo-NHS reaction was done to conjugate the small molecules to two identical DNA sequences with varying lengthes. Through the methods of ultraviolet-visibile-near infrared absorption spectroscopy, near infrared fluorescence spectroscopy, and high-performance liquid chromatography characterization and structural analysis were performed. The results showed the successful conjugation of the small molecules to the amino-modified DNA and an alteration in the corona phase. The small molecules were found to bind to the DNA at multiple locations and the length of the sequence was found to have an effect on the corona phase.<br>by Jasmine K. Harris.<br>S.B.
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Hwang, Sung Hee. "I. Combinatorial solid-phase synthesis of isoxasolinopyrroles. II. OBOC small molecule combinatorial library encoded by halogenated mass-tags /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2004. http://uclibs.org/PID/11984.
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Yamakawa, Tatsuya. "Screening of human cDNA library reveals two differentiation-related genes, HHEX and HLX, as promoters of early phase reprogramming toward pluripotency." Kyoto University, 2016. http://hdl.handle.net/2433/217142.
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Ruda, Marcus. "Design and synthesis of steroid mimetic libraries using solid phase techniques /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-049-4/.
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Huang, Adela Ya-Ting. "Advancing dendrimer synthesis : solid-phase and self-assembly approach." Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0146.
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Les dendrimères sont très prometteurs du fait de leur structure unique et de leur multivalence. Cependant, leur synthèse souffre de problèmes de défauts de structure et de présence de produits secondaires très similaires. Des approches synthétiques alternatives sont donc fortement désirées. L'objectif de ma thèse consiste à explorer la synthèse sur support solide et l’approche d'autoassemblage pour la préparation de dendrimères.La première partie de ma thèse se concentre sur la synthèse de dendrimères en phase solide. Nous avons tout d'abord développé une méthode de synthèse pour les dendrimères poly(amidoamines) basée sur la chimie des peptides. Nous avons ensuite construit une petite bibliothèque de dendrimères de type triazine en faisant varier la taille et la terminaison des dendrimères pour créer une variété de dendrimères. Nous avons aussi tenté de synthétiser des dendrimères poly(aminoesters) bien que nous n'ayons pu les obtenir du fait du caractère labile de ces dendrimères.La deuxième partie de ma thèse vise à appliquer l’approche d'autoassemblage pour la construction de dendrimères supramoléculaires comme théranostiques combinant l'imagerie et la thérapie. Nous avons synthétisé un petit dendrimère amphiphile portant DOTA pour chélater le Gd (III). Ce dendrimère est capable de s'autoassembler en supramolécule et d’encapsuler l’agent anticancéreux doxorubicine, pour construire des agents théranostiques à base de dendrimères multivalents.L’ensemble de ma thèse se consacre au développement de stratégies en phase solide et de l'autoassemblage pour construire des dendrimères pour les applications dans les domaines biomédicaux et des matériaux<br>Dendrimers hold great promise for wide applications thanks to their unique structural architecture and multivalent cooperativity. However, dendrimer synthesis often suffers from structural defects caused by incomplete reactions and difficulties associated with purification. Consequently, alternative synthetic approaches to overcome the limitations of current dendrimer synthesis are in high demand.My first PhD project mainly focuses on establishing novel strategies and methodologies for solid-phase dendrimer synthesis with advantages of convenient complete synthesis and easy purification procedures. We first developed a new and concise solid-phase synthesis of PAMAM dendrimers based on the adoption of peptide synthesis chemistry. We then constructed a small library of triazine dendrimers varying in generations and surface groups with a view to rapidly synthesizing dendrimers with structural diversity. We also strived to synthesize poly(aminoester) dendrimers although we had difficult to get it thorough.My second PhD program aims to apply the self-assembly approach for constructing supramolecular dendrimer theranostics. A small DOTA-conjugated amphiphilic dendrimer with Gd(III)-chelation was synthesized and self-assembled into supramolecular nanomicelles to encapsulate the anticancer drug doxorubicin. The obtained system constitutes a multivalent nanotheranostic to combine imaging purpose with therapeutic utility.In summary, my PhD program mainly contributes to elaborating strategies for dendrimer synthesis using both solid-phase method and self-assembly approach in the view to realizing and broadening their applications in the arenas of biomedical and material sciences
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Chen, Xingguo Ronald. "Pin1 Catalytic and WW Domain Ligands." Diss., Virginia Tech, 2011. http://hdl.handle.net/10919/37824.
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Pin1 is a peptidyl prolyl isomerase (PPIase) enzyme with two domains, the catalytic domain and the WW domain. Both domains specifically bind pSer/pThrâ Pro motifs. Pin1 plays an important role in regulating the cell cycle, and it is involved in many diseases, such as cancer, HIV-1, Alzheimerâ s disease, asthma, hepatitis B, and rheumatoid arthritis. Pin1 is a very promising target for new drug development.
Three stereoisomers: (R,S)-, (S,R)- and (S,S)-Acâ pSerâ Ψ[(Z)CH=C]â Pipâ 2-(2-naphthyl)ethylamine were synthesized as inhibitors binding to the Pin1 catalytic domain. The (R,S)- and (S,R)-isomers were synthesized via a 13-step route, with overall yields of 2.0% and 1.4%, respectively. The newly formed stereogenic center in the piperidyl ring was introduced by a Luche reduction, followed by a stereoselective [2,3]-Still-Wittig rearrangement. The configuration of the stereocenter was determined by NOESY of a bicyclic derivative. The (Z)- to (E)-alkene ratio in the rearrangement was (5.5:1). The (S,S)-isomer was obtained as the epimerized by-product resulting from the (S,R)-isomer in the Na/NH3 deprotection step. The IC50 values for Pin1 inhibition were: 52, 85, and 141 μM, respectively. We concluded that in this Z-alkene isostere, the R-configuration would be preferred at both stereogenic centers, as mimics of L-Ser and L-Pip, to improve the affinity.
Combinatorial chemistry is a powerful method to discover biologically active compounds, and solid-phase synthesis is most commonly used to synthesize combinatorial libraries. To identify ligands for the Pin1 WW domain, a library, R1COâ pSerâ Proâ NHR2, was designed. A new solid-phase phosphorylating reagent (SPPR) containing a phosphoramidite function was synthesized in one step from commercially available Wang resin. The SPPR was applied in the preparation of a designed library through parallel synthesis. The library contained 357 members (17 à 21), and was screened by an enzyme-linked enzyme binding assay (ELEBA). The best hits were resynthesized, and the competitive dissociation constants, Kd-rel, were measured by ELEBA, with a Kd-rel value of 130 μM for the best ligand. The absolute dissociation constants will be measured by our collaborator, Prof. Jefferey Peng, University of Notre Dame, using NMR methods. Besides the identification of the Pin1 WW domain ligands, I created a practical method for solid-phase synthesis of phosphopeptides.<br>Ph. D.
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Nilsson, Jonas. "Design, Synthesis and Characterization of Small Molecule Inhibitors and Small Molecule : Peptide Conjugates as Protein Actors." Doctoral thesis, Linköpings universitet, Organisk Kemi, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-3943.
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This thesis describes different aspects of protein interactions. Initially the function of peptides and their conjugates with small molecule inhibitors on the surface of Human Carbonic Anhydrase isoenzyme II (HCAII) is evaluated. The affinities for HCAII of the flexible, synthetic helix-loop-helix motif conjugated with a series of spacered inhibitors were measured by fluorescence spectroscopy and found in the best cases to be in the low nM range. Dissociation constants show considerable dependence on linker length and vary from 3000 nM for the shortest spacer to 40 nM for the longest with a minimum of 5 nM for a spacer with an intermediate length. A rationale for binding differences based on cooperativity is presented and supported by affinities as determined by fluorescence spectroscopy. Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (HSQC) spectroscopic experiments with 15N-labeled HCAII were used for the determination of the site of interaction. The influence of peptide charge and hydrophobicity was evaluated by surface plasmon resonance experiments. Hydrophobic sidechain branching and, more pronounced, peptide charge was demonstrated to modulate peptide – HCAII binding interactions in a cooperative manner, with affinities spanning almost two orders of magnitude. Detailed synthesis of small molecule inhibitors in a general lead discovery library as well as a targeted library for inhibition of α-thrombin is described. For the lead discovery library 160 members emanate from two N4-aryl-piperazine-2-carboxylic acid scaffolds derivatized in two dimensions employing a combinatorial approach on solid support. The targeted library was based on peptidomimetics of the D-Phe-Pro-Arg showing the scaffolds cyclopropane-1R,2R-dicarboxylic acid and (4-amino-3-oxo-morpholin-2-yl)- acetic acid as proline isosters. Employing 4-aminomethyl-benzamidine as arginine mimic and different hydrophobic amines and electrophiles as D-phenylalanine mimics resulted in 34 compounds showing IC50 values for α-thrombin ranging more than three orders of magnitude with the best inhibitor showing an IC50 of 130 nM. Interestingly, the best inhibitors showed reversed stereochemistry in comparison with a previously reported series employing a 3-oxo-morpholin-2-yl-acetic acid scaffold.
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Subramaniam, Raja. "Simplified Routines for Sample Preparation and Analysis of Chemical Warfare Agent Degradation Products." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-54639.
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The thesis describes the development of new and improved methods for analyzing degradation markers from organophosphorus Chemical Warfare Agents (CWAs). Paper I and II describes an innovative and significantly improved method for the enrichment, derivatization (trimethysilylation) and GC-MS analysis of a broad range of organophosphorus CWAs degradation markers, namely the alkylphosphonic acids and a zwitterionic compound. That was achieved using solid phase disc extraction in combination with solid phase derivatization. The new method overcomes most limitations observed with existing techniques: it offers almost 100 % recoveries, requires no elution or evaporation steps, facilitates miniaturization of the solid sorbent and reagent, is compatible with in-vial derivatization, and minimizes the chromatographic background due to the use of a highly selective anion exchange sorbent disc. Paper III describes the development of new fluorinated diazomethane derivatization reagents and their evaluation for rapid and high sensitivity screening and identification of nerve agent degradation markers. The reagents are water-tolerant to some extent, which simplifies the derivatization step. The best reagent identified was 3,5-bis(trifluoromethyl)benzyl diazomethane, which outperformed the other reagent isomers tested and also the established commercial alternative, pentafluorobenzylbromide, allowing for the rapid (5 min) and direct derivatization of a 25 μL aqueous sample in acetonitrile. The spectra of the formed derivatives (high-energy collision induced fragmentation MS/MS) were used to construct a database (Paper IV) that proved to be superior in terms of match factor and probability compared to EI data gathered for trimethylsilyl derivatives. The study also focused on efforts towards achieving detailed structure information on the alkyl chains of the compounds in question using diagnostic ion interpretation. The final paper (paper V) describes the first rapid direct derivatization method for analyzing nerve agent metabolites in urine at trace levels. The method is based on the derivative from the paper III and the unambiguous identification was proven using a combination of low resolution and high resolution negative ion chemical ionization selected ion monitoring techniques. Novel results presented in these papers include: the first in-situ derivatization of alkylphosphonic acids on an SPE disc; the first direct derivatization of nerve agent markers in water and biomedical samples; the first high sensitivity GC-MS screening for these markers; and the first highly reproducible high-energy isomer specific CID MS/MS library. Overall, the results presented in this thesis represent significant contributions to the analysis of nerve agent degradation products.
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Aftab, Obaid. "Towards High-Throughput Phenotypic and Systemic Profiling of in vitro Growing Cell Populations using Label-Free Microscopy and Spectroscopy : Applications in Cancer Pharmacology." Doctoral thesis, Uppsala universitet, Cancerfarmakologi och beräkningsmedicin, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-234565.
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Modern techniques like automated microscopy and spectroscopy now make it possible to study quantitatively, across multiple phenotypic and molecular parameters, how cell populations are affected by different treatments and/or environmental disturbances. As the technology development at the instrument level often is ahead of the data analytical tools and the scientific questions, there is a large and growing need for computational algorithms enabling desired data analysis. These algorithms must have capacity to extract and process quantitative dynamic information about how the cell population is affected by different stimuli with the final goal to transform this information into development of new powerful therapeutic strategies. In particular, there is a great need for automated systems that can facilitate the analysis of massive data streams for label-free methods such as phase contrast microscopy (PCM) imaging and spectroscopy (NMR). Therefore, in this thesis, algorithms for quantitative high-throughput phenotypic and systemic profiling of in vitro growing cell populations via label-free microscopy and spectroscopy are developed and evaluated. First a two-dimensional filter approach for high-throughput screening for drugs inducing autophagy and apoptosis from phase contrast time-lapse microscopy images is studied. Then new methods and applications are presented for label-free extraction and comparison of time-evolving morphological features in phase-contrast time-lapse microscopy images recorded from in vitro growing cell populations. Finally, the use of dynamic morphology and NMR/MS spectra for implementation of a reference database of drug induced changes, analogous to the outstanding mRNA gene expression based Connectivity Map database, is explored. In conclusion, relatively simple computational methods are useful for extraction of very valuable biological and pharmacological information from time-lapse microscopy images and NMR spectroscopy data offering great potential for biomedical applications in general and cancer pharmacology in particular.
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Leslie, Susan Elder. "An examination of the information behaviour of new entrepreneurs in the start-up phase of a business submitted to the School of Information Management, Victoria University of Wellington in partial fulfilment of the requirements for the degree of Master of Library and Information Studies /." ResearchArchive@Victoria e-Thesis, 2009. http://hdl.handle.net/10063/1271.
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Joo, Sang Hoon. "Synthesis and screening of support-bound combinatorial cyclic peptide and free C-terminal peptide libraries." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1195561420.
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LIN, MING-CHUN, and 林明君. "Ractopamine Detection Through Phage Display Peptide Library." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/48211792124612562148.
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Chou, Ying-Hsiu, and 周盈秀. "Construction of human phage-displayed scFv library." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/13301253686668832243.
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碩士<br>國立成功大學<br>醫學檢驗生物技術學系碩博士班<br>97<br>Antibodies are applied not only for diagnosis, but also in research and
clinical treatments. In order to generate high-affinity monoclonal antibodies, three
techniques have been developed including hybridoma engineering, transgenic mice
and phage-displayed antibody libraries. Although the hybridoma technology is the
major tool for monoclonal antibody production, it still has some problems. For
example, the methodology cannot be curable directly in human disease. Hence, the
phage antibody display system has become more attractive to scientists. We
attempted to develop a na��ve human single chain antibody (scFv) library by phage
display system. We first extracted total RNA from the leukocytes of peripheral
blood from 290 volunteers. The heavy chain and light chain cDNA fragments were
synthesized by reverse transcription reaction. Thirty-one forward and reverse
primers were used for the cloning of heavy chain variable regions (VH) and light
chain variable regions (VL) fragments from the cDNA fragments. The cloned heavy
and light chain variable regions were amplified and linked with linkers by
overlapping PCR. The linkers encoded the 15 amino acids (Gly4Ser)3 that have
overhangs of perfect complementarity with VH and VL genes. The purified scFv
fragments were ligated to the phagemid pIGT5 for electroporation into Escherichia
coli HB2151 competent cells. In order to increase the binding avidity of scFv
during bio-paining, we had constructed pIGT5 phagemid which expressed trypsin
and enterokinase cutting site between pIII and scFv. We also established Ex-phage
in which the pIII protein could not be synthesized in non-amber suppressed strain
owing to the amber codon before pIII. Hence, the binding phage can be released
after enzymatic digestion and amplified for next paining. After the library
constructed, we will apply this antibody library to discover new carbohydrate
antibodies.
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Lin, Shu-Jian, and 林淑娟. "Construction of scFv Phage Display Library Against Diphenylamine." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/92251249392714967412.
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碩士<br>國立中興大學<br>生命科學院碩士在職專班<br>92<br>Diphenylamine is an antioxidant, having the skeleton structure of non-steroidal anti-inflammatory drugs. In the use of agriculture, it is as a dipping solution to prevent superficial scald in the surface of apple.
Diphenylamine also is a fungicide and was used in the pesticides with high concentration. It is an important derivative of BXT, and often used in hair color dye. It is toxic, possible mutagen and teratogen, and harmful in contact with skin.
Because diphenylamine is a hapten, in this experiment, we use conjugate reagent to synthesize the bovine serum albumin with diphenylamine together as an immunogen. After immunization, and confirmed with ELISA, we take out the spleen of mouse, and use a series of degenerate primers covering recently known murine immunoglobulin gene family and performing RT-PCR to amplify variable region of heavy chain (VH) and light chain (VL) gene.
The amplified VH and VL DNA fragments were assembled to perform single chain fragment of variable region (scFv). The scFv DNA were cloned to the pHEN2 vector, and then transformed to E. coli. We use the technique of phage display system to construct the library of scFv for further selection. The constructed antibody phage display library was confirmed the diversity by BstNI restriction fingerprinting analysis.
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Yi-WenChang and 張憶文. "Discovery of antibodies by phage display human scFv antibody library." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/15750785477965469165.
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Hu, Ting-Yuan. "Titanium-binding Peptide Isolated from Phage-displayed Random Peptide Library." 2007. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-3007200716354000.
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Hu, Ting-Yuan, and 胡庭元. "Titanium-binding Peptide Isolated from Phage-displayed Random Peptide Library." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/93998814871702072948.
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碩士<br>臺灣大學<br>臨床牙醫學研究所<br>95<br>Since the introduction of titanium root-form dental implant in 60’s, it has become a widely used treatment modality in rehabilitation of completely or partially edentulous patients. However, clinicians still face the failure of implant treatment or its complication, such as progressive marginal bone loss, recession of marginal mucosa, or absence of osseointegration, inspite of its reported high success and survival rates in the literatures. In late 90’s, the application of biomimetic modification has been introduced to overcome the problems because of the advances in the understanding of the biology of bone healing process at molecular level. However, there is only marginal success in this field of research after a decade of efforts. One of the obstacles to the success is lack of an efficient and controllable coating methodology to achieve an biologically active surface with diverse function required for the healing process of surrounding tissues. In this study, we identified small adaptor peptides with titanium-binding property with a powerful screening methodology, phage display system. By five rounds of biopanning on Ti-6Al-4V discs, we isolated a clone of phage PT3 with high affinity to the surface of titanium, as verified by ELISA. We also conjugated PT3 at the amino terminus of hBMP-2 and expressed the fusion protein in E.coli. The identity of expressed protein was confirmed with mass spectrophotometry. It was then refolded and purified for further application in evaluation of the effect of immobilized growth factors on the healing process. This novel strategy may offer us a clinically efficient and easy-to-apply coating method in implant treatment.
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huang, chi hung, and 黃濟鴻. "Generation of scFv against Thalidomide stereoisomer from phage display library." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/65105760400880058834.
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碩士<br>國立中興大學<br>生物化學研究所<br>91<br>Thalidomide was first introduced in the 1950s as a sedative drug. Because it was deemed to be safe, it was prescribed for nausea and insomnia in pregnant women. However, it was found to cause the severe birth defects in children whose mothers had taken the drug in their first trimester of pregnancy. This was a terrible tragedy. Deepgoing research realized that one of thalidomide enantiomer possesses the desired therapeutic effect of quelling nausea, but its mirror compound produces fetal deformities.
In this work, we synthesis thalidomide conjugated with BSA as an immunogen in order to produce related antibodies. After immunization, RT-PCR was performed and a series of primers was used to amplify splenic VH/VL genes. Then, amplified VH/VL were assembled to construct the antibody phage display library against thalidomide. The constructed antibody phage display library was being selected against immunogen after comfirming the diversity by BstNI restriction enzyme fingerprinting analysis. After three rounds of selection, we have isolated two phage clones with weak affinity against (+) or (-) thalidomide individually. Colony PCR and western blotting analysis showed these two clones bear full-length of scFv DNA fragment and are able to express scFv. Competitive ELISA assay also confirmed the specificity of these two scFvs against (+)-thalidomide or (-)-thalidomide respectively.
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Hsia, Ko-Chiang, and 夏可強. "Isolation and characterization of lipase abzyme from phage displayed antibody library." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/mcyy49.
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碩士<br>國立東華大學<br>生物技術研究所<br>94<br>Interactions of antigen and antibody resemble to some extents the recognition and specific binding between enzyme and substrate. For this reason, antibodies may have certain enzymatic activity and were named abzymes or catalytic antibodies. In the present work we aimed to get lipolytic abzymes through the selection from phage displayed antibody library. A transition state analog for selection of lipolytic abzymes was synthesized. The structure composed of 4-nitrophenyl activated phosphonate, the transition state analog, (TSA) of lipases/esterases, connected to a biotin moiety through a 15 carbon chain spacer. After several rounds of selection, we obtained six monoclonal phage particles capable of binding significantly to the TSA. However, no apparent enzyme activity was observed using the solution of these phage particles for lipase enzymatic assay. Then, we used strain HB2151 (K12 ara Δ(lac-proAB) thi/F' proA+B lacIq lacZΔM15) to express the soluble antibody fragment. Through the His-tag purification, the purified antibody protein was examined further for its binding capability to TSA and the possible enzymatic activity. The present method presents a more efficient and convenient means to find new abzyme. Catalytic antibodies may have the potential to replace current enzymes and may have very diverse application.
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"Applications of phage-displayed antibody library for antibody discovery and engineering." Thesis, 2008. http://library.cuhk.edu.hk/record=b6074647.
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Antibodies are one of the most useful molecules with affinity of binding and specificity for in vitro and in vivo diagnosis, or for immunotherapy of human diseases. In recent years, phage-displayed antibody library has been widely adopted to select tailor-made antibodies in a fast, high-throughput mode, as an alternative of traditional hybridoma technology. Although phage display has been introduced for about 20 years, the applications and development of this technology still have a rich space to be explored.<br>Attempts are made in the present study to extend three applications of the phage displayed antibody library in antibody discovery and engineering. Firstly, a CDR3-randomized phage-displayed scFv library was constructed from genomic DNA of mouse. Following biopanning, anti-peptide of mas oncoprotein scFvs were isolated and identified. These results illustrate the potential use of the genomic phage-displayed library for anti-peptide antibodies selection. Secondly, we described the isolation of anti-idiotypic scFvs against a chimeric anti-CD22 mAb from an immunized phage-displayed scFv library. The isolated anti-Id scFvs were able to capture the immune response of chimeric anti-CD22 mAb with high specificity. This reagent will enhance our understanding of the therapeutic mechanism of anti-CD22 mAb in non-Hodgkin's lymphoma treatment, and may be applied to probe the pharmacokinetics, tissue distribution, and modulation of anti-CD22 mAb in vivo.<br>In conclusion, we have attempted various approaches to identify specific anti-peptide scFvs, anti-idiotypic scFvs and passive anti-tumor scFvs. These results extend the applications of phage display technology in antibody discovery and engineering.<br>Our approach enables us to isolate selective and sensitive anti-idiotypic antibodies and could be exploited for other antibodies with clinical and biological applications. Thirdly, we profile a strategy to select and identify markers on tumor cell surface using phage-displayed antibodies from mice bearing xenograft tumor. Our data imply that passive antibodies in cancer patients may be obtained from the immune repertoire of cancer patients. Besides, we found a cell surface antigen was up-regulated more than 3-fold in mas-expressing cells. We further use the targeting antibody to construct a tumor endoprotease-activated immunotoxin.<br>Zhao, Qi.<br>Adviser: Wing-Tai Cheung.<br>Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3499.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2008.<br>Includes bibliographical references (leaves 227-250).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstracts in English and Chinese.<br>School code: 1307.
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Tseng, Sy-Woei, and 曾斯偉. "Construction of phage-display peptide library for the screening of antibody epitope." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/04418277385769813527.
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碩士<br>國立中興大學<br>分子生物學研究所<br>88<br>Abstract
Phage display technology, consisting of the expression of target sequence in the surface of phage particles and the affinity selection, is a biological system that facilitates the cloning and rapid selection of peptides or proteins from large combinatorial libraries. Libraries are generated by cloning of a batch of DNA encoding millions of variants of certain ligands into the phage genome or phagemid as a fusion to the gene encoding one of the phage coat proteins. Upon expression, the fusion coat protein is incorporated into new phage particles that are assembled in the bacterium and consequently presented on the phage surface, with its genetic material residing within the phage particles. The purpose of this study is to construct a random peptide library containing both linear and conformational epitopes to serve as an “all purpose” peptide source for searching of disease-related peptides. To achieve this goal, EcoRI-HindIII DNA fragments encoding 45 amino acid residues, of which 36 have random sequences, were cloned into the corresponding sites of the modified pCANTAB5E plasmid and the resulted plasmids were transformed into E. coli TG1. The percentage of insert-containing colonies in the transformants was evaluated by PCR using primers flanking the insert DNAs. Approximately, 83% of the clones contained inserts. The expression of the insert gene was determined by colony immunoblot analysis of a portion of the original clones using the antibody specific for E-Tag which is located downstream of the insert. The number of clone capable of expression coincided with the percentage of the insert-containing clones. Finally, the diversity of the library was evaluated by sequencing of 10 randomly picked clones. The results indicated that all exhibited difference sequences. The diversity of the library was estimated to be 4.4 x 106 according to the above information,. To establish a system for identification of disease-related peptides, affinity selection was performed using sera from system lupus erythematosus patients.
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Ying-HsiuLiu and 劉盈秀. "Selection and characterization of EV71 antibody by phage display human scFv antibody library." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/42895278044035675388.
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碩士<br>國立成功大學<br>醫學檢驗生物技術學系碩博士班<br>101<br>Enterovirus 71 (EV71) which causes hand-foot-mouth disease (HFMD) and herpangina belongs to the Picornaviridae family with ss(+) RNA virus. In 1998, EV71 outbreak in Taiwan and resulted in 78 fatal cases of 405 severe complications related neurological or cardiopulmonary symptoms. EV71 has been threatened the children under 3 years of age in Asia-Pacific area. Vaccination of EV71 seems the most efficient to eradicate the infectious disease. However, the development of vaccines is still in clinical trials. Antibodies also develop for early detection and diagnosis to control and prevent EV71 transmission. Different antibody format can be expressed by phage display technology via genetic engineering. Here, we selected the phage clones to against EV71 from the human scFv antibody library via biopanning. Procedure of biopanning included binding the target, washing out unbound phage and amplification of binders. Phage clones in total 385 were random picked and grouped by RFLP with SalI restriction enzyme. Five groups were identified and sequenced the nucleotides. Sequence data were aligned with the international ImMunoGeneTics information system (IMGT). We found that all of five phage clones contain a variable heavy sequence. Phage clone 3 and 9 contained full length scFv nucleotide sequence but formed no functional scFv antibody in protein level. However, the phage clones 6 and 13 showed binding ability to against EV71 that revealed in ELISA results. In addition, the phage clone 6 and 13 can recognize EV71virus particles and VP2 protein in western blot. Phage clone 6 and 13 detected EV71 in RD cell can be observed by immunofluorescence assay (IFA). In this study, the data suggested that phage clone 6 and 13 from the human scFv antibody library can be applied for EV71 research or diagnosis.
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Huang, Shih-Tsung, and 黃仕聰. "Development of Fully Human Anti-HDGF Antibody from Phage-Displayed Human Naïve ScFv Library." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/92wr6a.
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博士<br>國立中山大學<br>海洋生物科技博士學位學程<br>107<br>Hepatocellular carcinoma (HCC), a deadly disease worldwide. For advanced HCC therapy, Sorafenib is the only conventional drug, however, low response rate, drug resistance, and strong side effects had been reported in Sorafenib therapy. Therefore, investigation of novel therapeutic strategy is a critical issue. HDGF is a cancerous factor in various cancer types including HCC. To develop anti-HDGF neutralizing antibody, phage display technology provides an effective in vitro selection strategy to identify fully human antibody. In the biopanning, thirteen HDGF affinity phage clones had been identified from phage display human naïve scFv library using unique Fc fusion protein/protein G dynabeads method. After fully human IgG construction and production, these antibodies were further confirmed binding affinity using ELISA, immunofluorescence, and HDGF conditional knockdown Huh7 cell line. Among them, hmAb-7 exhibited multiple anti-cancer activity, including cell growth, colony formation, invasion and sphere formation in Huh7, HepG2, and SK-Hep1 cells. The anti-cancer effects of hmAb-7 might participated with downregulation of PI3K/AKT/mTOR signaling pathway. The hmAb-7 inhibits of invasion through MMP-2 down-regulation; sphere formation was eliminated through downregulation of cancer stemness genes. In continuously cultured Huh7 and HepG2 cells, these cells present higher colony and spheroid forming features, however, the cancerous behavior able to abolish through hmAb-7 treatment. In sorafenib resistant Huh7 cells, hmAb-7 presents colony and sphere inhibition ability. Furthermore, the Huh7 xenograft model in NOD/SCID mice was performed and the data indicated that hmAb-7 slightly inhibited tumor growth (p = 0.1091) and prolong the overall survival for 14 days. Although anti-HDGF hmAb-7 single treatments not significantly inhibit tumor growth in vivo, but based on the cancerous inhibition effects in vitro, combination therapy might provide an approach to enhance therapeutic effect. Furthermore, a HDGF sandwich ELISA platform with well linear range between 10-0.15625 ng/ml has been established and able to detect the HDGF level in human and rat serum. Anti-HDGF antibody was also used for extended applications, included in analgesic application and allow to treat cancer stemness and Sorafenib drug resistant Huh7 cells (Huh7-SR). Our finding indicated anti-HDGF hmAb-7 exhibited analgesic effect in rat chronic constrictive injury (CCI) model plus with Adenoviral HDGF gene delivery. Besides, hmAb-7 not only shown sphere and colony inhibitory effects in continuous spheroid cell cultured Huh7 and HepG2 cells, but also inhibited cancerous behaviors in Huh7-SR cells. Finally, adenovirus mediated anti-HDGF-scFv gene delivery suppressed the colony and sphere formation in both Huh7 and Huh7-SR cells. The adenovirus mediated scFv gene delivery provided a novel approach for anti-HDGF therapy and allowed for further basic studies.