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.