In Vitro Selection Of DNA Aptamer Binding To TNF And Function Research Of The Aptamer

Human tumor necrosisi factor a (hTNF-a) is one of the most important inflammatory cytokines that acts as a mediator in inflammatory and immunologic reactions and plays a key role in host defenses against infection. The over expression of hTNF-a can lead to serious inflammation effects, such as shock, hypotension, thrombus, septicemia and even death. It has been implicated in many autoimmune and inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, chronic heart failure and septic shock. It is important to inhibit the bio-activity of hTNF-a for the treatment of some inflammatory diseases.Compared with traditional recombinant protein drugs, small molecular drugs have any advantages, such as easy absorbance, no immunity and high affinity etc. To obtain oligonucleotide aptamers, specifically binding to TNF, and to find the relationship between the structures and affinities, and to determined whether the aptamers can be used as a novel molecule to anti-TNF, a synthetic 71 mer random DNA library was subjected to 15 rounds of selection by using SELEX (systematic evolution of ligands by exponential enrichment) protocol against TNF. The selected aptamers were cloned and sequenced. Software package DNASYSv2.5 is employed to analyze the conserved sequences and second structures of aptamers, respectively. Affinities of aptamers to TNF were visualized by biotin-streptavidin-horseradish peroxidase system. The L929 cells were used to certify the anti-TNF nature of aptamers. The results show that we have got the special high affinity aptamers of TNF. These works laid a foundation for further research and clinical investigation.