magistrsko delo
Laura Vuga (Author), Tomaž Bratkovič (Mentor)

Abstract

V tem delu so opisani postopki načrtovanja ter optimizacije peptidnih ligandov Fc regije imunoglobulinov G. Peptidne ligande smo izrazili na bakteriofagnem nosilcu v obliki monovalentne predstavitve. Opisani so postopki transformacije, gojenja ter izolacije rekombinantnih fagmidnih delcev. Relativno afiniteto različic peptidnih ligandov do humanih IgG smo določili z encimskoimunskim testom. Optimizirali smo sestavo pufrov za spiranje, blokiranje ter redčenje vzorcev, nato pa tudi testirali različne titre fagov, na podlagi česar smo določili optimalne pogoje, ki smo jih uporabili pri nadaljnjih testih. Fagmid, na katerem smo peptidne ligande izražali, smo tekom dela spremenili ter optimizirali. S tem smo uspeli precej znižati vrednosti nespecifične adsorpcije fagov, kar je znatno izboljšalo resolucijo rezultatov. Na koncu smo tako pridobljene peptidne ligande primerjali s starševskim peptidom in na podlagi rezultatov izvedli analizo povezave med strukturo in aktivnostjo (oz. afiniteto; angl. structure-activity relationship, SAR). Starševski peptid smo uspeli optimizirati, analiza SAR pa nam je dala izsledke, ki bodo uporabni za nadaljnje raziskave ter optimizacijo alternativnih ligandov Fc regije IgG.

Keywords

protitelesa;fagi;E. coli;ELISA;peptidni ligandi;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FFA - Faculty of Pharmacy
Publisher: [L. Vuga]
UDC: 602.68:602.641(043.2)
COBISS: 78963203 Link will open in a new window
Views: 225
Downloads: 31
Average score: 0 (0 votes)
Metadata: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Other data

Secondary language: English
Secondary title: Establishing the model of the relationship between the structure and the affinity of the peptide ligand of immunoglobulin G Fc region
Secondary abstract: This thesis describes the design and optimization of peptide ligands of the immunoglobulin G Fc region. Peptide ligands were displayed on a bacteriophage vector in the monovalent setting. The protocols of transformation, cultivation and isolation of phagemid particles are described herein. The relative affinities of peptide ligand variants to human IgG were determined by enzyme-linked immunosorbent assay. We optimized the composition of washing, blocking and dilution buffers. We tested different phage titers, on the basis of which we determined the optimal conditions, that we used in the following tests. We also changed and optimized the original phagemid on which the peptide ligands were displayed. This way, we were able to significantly reduce the nonspecific phage adsorption, that was previously distorting the results. Finally, the peptide ligands thus obtained were compared with their parent peptide and structure-activity (affinity) relationship (SAR) analysis was performed based on the results. We were able to optimize the parent peptide, and the results can be used for further research and optimization of alternative ligands of IgG Fc region.
Secondary keywords: antibodies;phages;peptide ligands;
Type (COBISS): Master's thesis/paper
Study programme: 0
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Pages: XII, 52 f.
ID: 13620771