Izbor peptidnih ligandov stafilokoknega proteina A iz bakteriofagnih peptidnopredstavitvenih knjižnic : [diplomska naloga]

Masilo, Katja (author), Lunder, Mojca (mentor)

Stafilokokni protein A je protein, ki ga na svoji površini izraža bakterija Staphylococcus aureus. Z vezavo konstantne regije protiteles človeškega ali živalskega izvora bakteriji omogoča izogibanje pridobljenemu imunskemu odzivu gostitelja. Zaradi učinkovitih mehanizmov izogibanja imunskemu sistemu in odpornosti na številne antibiotike, znanstveniki iščejo nove načine zdravljenja stafilokoknih okužb. Ena izmed možnih strategij je ciljana dostava učinkovine do proteina A s pomočjo liganda, ki se veže nanj. Protein A je tudi široko uporaben v biotehnologiji kot imunokemijski reagent za identifikacijo ter čiščenje protiteles. V diplomski nalogi smo s pomočjo afinitetnih selekcij na streptavidinu in proteinu A iz dveh bakteriofagnih knjižnic poskušali izselekcionirati peptidne ligande, ki se vežejo na streptavidin oziroma protein A. V testni selekciji na streptavidinu smo izolirali klone z značilnim motivom HPQ z visoko afiniteto do streptavidina. V afinitetni selekciji na proteinu A z nespecifično elucijo smo iz knjižnice Ph.D.-C7C™ izolirali peptid CAKTDPRGC in s kvantitativno verižno reakcijo s polimerazo v realnem času potrdili specifičnost vezave na protein A. Peptid ima zelo podobno zaporedje kot konstantna regija protiteles, ki je naravni ligand proteina A. Selekcija iz knjižnice Ph.D.-C7C™ s specifično elucijo ni bila uspešna. V selekciji s specifično elucijo s konstantno regijo protiteles iz knjižnice Ph.D.-12™ pa smo izolirali peptide z izrazitim motivom GKL. S kvantitativno verižno reakcijo s polimerazo v realnem času smo potrdili vezavo peptidov GGKLTTIHLHST in GISDFMQTKMPL na protein A.

• Language: Slovenian
• Keywords: protein A stafilokokne okužbe bakteriofagne knjižnice peptidni ligandi afinitetna selekcija fagni kloni diagnostika
• Work type: Undergraduate thesis
• Typology: 2.11 - Undergraduate Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [K. Masilo]
• Year: 2013
• Number of pages: 60 str.
• PID: 20.500.12556/RUL-44271
• UDC: 543+547.96(043.2)
• COBISS.SI-ID: 3395697
• Publication date in RUL: 10.07.2015

Secondary language

• Language: English
• Title: Selection of ligands of staphylococcal protein A from phage-displayed peptide libraries

Abstract

Staphylococcal protein A is a surface protein of Staphylococcus aureus which binds human and animal antibody constant region to prevent adaptive immune response of the host. Due to effective mechanisms of evading the immune system and resistance to antibiotics, scientists are searching new ways of treating staphylococcal infections. One of possible strategies is protein A targeted drug delivery. Protein A is also widely used in biotechnology as an immunochemical reagent for antibody identification and purification. The aim of this research was to isolate peptide ligands with affinity to streptavidin and protein A from phage-displayed peptide libraries with affinity selection. With test affinity selection on streptavidin we isolated clones with typical motif HPQ and high affinity to streptavidin. With affinity selection on protein A from library Ph.D.-C7C™ with non-specific elution we isolated a peptide CAKTDPRGC and confirmed its binding to protein A with quantitative real-time polymerase chain reaction. The peptide's sequence is very similar to the sequence of antibody constant region, which is natural ligand of protein A. The selection from library Ph.D.-C7C™ with specific elution was not successful. We isolated peptides with distinct motif GKL with specific elution with antibody constant region from library Ph.D.-12™. Peptides GGKLTTIHLHST and GISDFMQTKMPL specifically bind to protein A, which was confirmed with quantitative real-time polymerase chain reaction.

• Keywords: Staphylococcal protein A Staphylococcus aureus phage-display protein A ligand affinity selection