Screeing DNA Aptamers Of Deoxynivalenol By SELEX

Deoxynivalenol (DON) is a kind of trichothecenes of the same chemical structure and biological activity produced by Fusarium, and is one of the least acutely toxic trichothecenes. It is a common contaminant of wheat and corn usually occurs in temperate climates that are particularly wet at the time of harvest in years. DON causes feed refusal, emesis, immunosuppression or immunostimulation, teratogeny, cytotoxicity, reproductive toxicity and human cancers. Therefore, a rapid and reliable method for the determination of deoxynivalenol is important to avoid the risk of DON consumption by humans and animals.SELEX (Systematic Evolution of Ligands by, Exponential Enrichment), is also known as a new selection technique in vitro. Aptamer, which is screened from the large random nucleic acids libraries by SELEX technique, is oligonucleotide that binds to targets with high affinity and specificity. As aptamer is resembled with antibody but superior to antibody for having a large number of target molecules, high affinity and specificity, stability and ease of production, regeneration. Aptamer has promising applications prospect in clinical therapy and laboratory diagnosis. This study aims to screen the aptamer with high affinity and specificity for cyclosporin A and set a base for establishing relative easy and quick laboratory detection methods.The study conjugated the DON to protein covalently at first. The study synthesized 87 bp single stranded DNA (ssDNA) random library containing 45 ranodm sequences flanked by invariant primer was subjected to 10 rounds of selection against DON-conjugated by SELEX method, which we applied biotin-streptavidin-horseradish peroxidase system to determine the binding affinity between the aptamers and DON. PCR products of the last round of selection were cloned and sequenced. Relative software was employed to analyze the primary structure and prediction secondary structure of the aptamers.After 10 rounds of selection and amplification that obtained the aptamers against DON. Meanwhile, we can see an increasing tendency of binding affinity between the aptamers and DON. The sequences of the 21 aptamer clones are 87 bp and the sequences are divided into 12 groups on the basis of primary sequence homology. The number 7 aptamer clones will be the focus of our study in future. They have steady configuration and high affinity. Hairpin loop are the main motif in prediction secondary structure which may be the binding structure between the aptamers and target peptides. Based on the selection of the aptamers against DON, we can set up relative analysis systems for Enzyme-Linked Oligonucleotide assay (ELOSA) and so on.