Optimizacija vezave aktivatorja receptorja TLR9 na peptide s strukturo ovite vijačnice

Lulić, Sanjin (author), Benčina, Mojca (mentor)

Nemetilirane molekule CpG-ODN aktivirajo prirojen imunski odziv z vezavo na receptor TLR9. Aktivacija prirojenega imunskega odziva pripelje do posledične aktivacije pridobljenega imunskega odziva in tvorbe imunskega spomina. V magistrskem delu smo pripravili polipeptidno ogrodje GFP:P3:P6:P10, na katerega smo v nadaljevanju želeli povezati aktivatorje receptorja TLR9 preko homolognih peptidov, ki tvorijo ovite vijačnice. Aktivatorje receptorja TLR9 smo kovalentno povezali s peptidom, ki tvori ovito vijačnico, pri čemer je izkoristek reakcije bil prenizek za nadaljnjo uporabo. Poleg tega smo za namen enostavnega čiščenja konjugata pripravili nosilec afinitetne kromatografije z magnetnimi kroglicami, ki je deloval na podlagi komplementarnosti molekul DNA. Po prvotno uspešni vezavi komplementarne molekule ODN na nosilec in spiranju le-te z nosilca, je kapaciteta nosilca izrazito padla.

• Language: Slovenian
• Keywords: sintezna biologija, prirojeni imunski odziv, receptor TLR9, peptidi, ovite vijačnice, kovalentno povezovanje
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [S. Lulić]
• Year: 2020
• PID: 20.500.12556/RUL-115185
• UDC: 601.4:577.27:602.6:579.8(043.2)
• COBISS.SI-ID: 9468793
• Publication date in RUL: 16.04.2020

Secondary language

• Language: English
• Title: Optimisation of TLR9 receptor activator conjugation to coiled coil peptides

Abstract

Unmethylated CpG-ODN molecules activate an innate immune response by binding to TLR9 receptor. The activation of the innate immune response results in the activation of adaptive immune response and thus also creating immune memory. In this MSc thesis we produced polypeptide GFP:P3:P6:P10 which acts as a scaffold for binding TLR9 receptor activators via coiled coil peptides. We have covalently bound activators of TLR9 receptors to the coiled coil peptides. The reaction had low yield and thus the conjugate was not suitable for further experiments. Furthermore we designed an affinity chromatography on the principle of complementary DNA molecules with magnetic beads for an easy and efficient cleaning of the produced conjugate. After the first successful binding of complement ODN molecule to the affinity carrier, the capacity of the carrier was significantly reduced.

• Keywords: synthetic biology, innate immune response, receptor TLR9, covalent coupling, peptides, coiled-coil peptides