Priprema adenovirusnih vektora za unos gena TLR5 u stanice pluća

master's thesis

• Title (english): Preparation of adenoviral vectors for insertion of the TLR5 gene
• Author: Vanessa Krasnić
• Mentor: Jelena Knežević (mentor)
• Mentor: Nada Oršolić (mentor)
• Committee member: Nada Oršolić (predsjednik povjerenstva)
• Committee member: Maja Matulić (član povjerenstva)
• Committee member: Maria Špoljar (član povjerenstva)
• Committee member: Jasna Lajtner (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2019-02-18, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Kronična opstruktivna plućna bolest (KOPB) i rak pluća značajni su uzročnici smrtnosti u svijetu. Izloženost duhanskom dimu predstavlja glavni čimbenik razvoja KOPB-a. Epidemiološka istraživanja pokazala su da je KOPB, neovisno o statusu pušenja, najveći čimbenik rizika za razvoj raka pluća. Genetička podloga povezanosti ovih patoloških entiteta je nepoznata. U održavanju homeostaze organizma važnu ulogu ima regulacija imunosnog odgovora. Bitnu ulogu u pokretanju imunosti imaju toll-like receptori (TLR) tako što prepoznaju patogenu pridružene slijedove i induciraju signalne puteve u domaćinu kao antimikrobni odgovor. Ranija istraživanja su pokazala da je genetička varijanta gena TLR5, N592S, povezana s nastankom KOPBa i raka pluća. Cilj ovog istraživanja je konstruirati replikacijski defektan adenovirusni vektor uvođenjem gena za TLR5wt i TLR5N592S u okosnicu adenovirusa tipa 5 (Ad5) koji bi omogućio unos tog gena u modelni organizam, u ovom slučaju humane stanice pluća (A549, WI38 i H1299), u svrhu daljnjih funkcionalnih istraživanja. Rekombinantni Ad5-TLR5 virus konstruiran je manipulacijom Ad5 genoma kao plazmida u E. coli korištenjem homologne rekombinacije u bakterijskom soju BJ5183. Ad5-TLR5wt i Ad5-TLR5N592S umnoženi su u humanim embrionalnim stanicama bubrega 293 (HEK293) i pročišćeni ultracentrifugiranjem u gradijentu cezijevog klorida.

Abstract (english)

Chronic obstructive pulmonary disease (COPD) and lung cancer are major causes of death in the world. Exposure to tobacco smoke presents the main factor in the development of COPD. Epidemiological research has shown that COPD represents, regardless of smoking status, the biggest risk factor for developing lung cancer. The genetic basis of the association of these pathological entities is unknown. The regulation of the immune response plays an important role in maintaining homeostasis. Toll-like receptors (TLR) are important for initiating immunity by recognising the sequences associated with the pathogen and inducing signal pathways in the host as an antimicrobial response. Previous researches have shown that the genetic variant of TLR5, N592S gene, is associated with COPD and lung cancer. The aim of this research was to construct a replication-defective adenoviral vector by introducing a gene for TLR5wt and TLR5N592S into the adenovirus type 5 (Ad5) backbone that would allow the insertion of this gene into a model organism, in this case human lung cells (A549, WI38 and H1299), for the purpose of further functional research. Recombinant Ad5-TLR5 virus was constructed by manipulating the Ad5 genome as a plasmid in E. coli using homologous recombination in bacterial strain BJ5183. Ad5-TLR5wt and Ad5-TLR5N592S were amplified in human embryonic kidney cells 293 (HEK293) and purified by ultracentrifugation in the caesium chloride gradient.

• Keywords: Kronična opstruktivna plućna bolest; rak pluća; gen TLR5; kloniranje; adenovirusni vektori
• Keywords (english): Chronic obstructive pulmonary disease; lung cancer; TLR5 gene; cloning; adenoviral vectors
• Language: croatian
• URN:NBN: urn:nbn:hr:217:157144
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2019-03-26 09:47:08