Screening Of DNA Aptamers Which Bind To Staphylococcal Aureus Enterotoxin B And Primary Application

[Objective]We have applied systematic evolution of ligands by exponential enrichment (SELEX) for screening single strand DNA(ssDNA) aptamers which specifically bind to Staphylococcal aureus enterotoxin B (SEB). Basing on a high affinity aptamer found, we used aptamers sandwich assay to detect SEB in patient′s serum samples, and we used this method to diagnose infection of Staphylococcal aureus. We explored the inhibitory effect of the aptamers on the superantigen activity of SEB in a Rat model.[Methods]1. The products of the enriched library were amplified, purified, TA cloned and cultivated in LB medium. Selection of single colony and extraction of plasmid was the first step. Then the ssDNA aptamers specifically binding to SEB were obtained through PCR amplification and separation by Dynabeads Carboxylic Acid.2. The affinity, specificity and other properties of aptamers were identified by direct assay, and aptamers were separately labeled digoxin, fluorescence and biotin.3. We respectively using the antibody-aptamer sandwich assay and library-aptamer sandwich assay to detect the quantitative of SEB. In the same time, we have compared the two detection methods. Then we using library-aptamer sandwich assay to detect the SEB levels in patient′s serum.4. The Rats were randomly divided into four groups, given different interventions and treatment. Subsequently we detected the concentration of ALT, AST, IL-4, IL-6, creatinine and uric acid in Rats serum. The results of each group were analysised by related statistical software. [Results]1. After cloning and electrophoretic analysis, thirty correct sequences were found, and get one aptamer with high affinity and specificity.2. Library-aptamer sandwich assay and antibody-aptamer sandwich assay could be used to detect the quantitative of SEB, and the results from two methods showed no statistical difference. The results from healthy control and bacterial infection group showed significant difference with SEB content in serum samples.3. The superantigen activity of SEB can be blocked in vivo by aptamers in a Rat model, and aptamers can inhibit the secretion of IL-4 and IL-6, and also can reduce the levels of AST, ALT, uric acid and creatinine in serum.[Conclusions]In the present study, the ssDNA aptamers specifically binding to SEB had been selected, and the library-aptamer sandwich assay of detection SEB in serum samples had been successfully established. Aptamer was similar to the role of antibodies, can be combined with SEB specifically, can serve potentially as a diagnostic molecule, and inhibit the biological activity of SEB. The aptamers specifically binding to SEB provide a foundation for the investigation and therapy of infection with Staphylococcal aureus.