In Vitro Selection Of Aptamers To Staphylococcus Enterotoxin A (SEA) And Their Application

【Objective】To screen functional aptamers binding to the Staphylococcus enterotoxin A (SEA)with high affinity and specificity through Systematic Evolution of Ligands byExponential enrichment (SELEX) and lay a foundation for the rapid diagnosticmethod to detect SEA and provide a new idea for the treatment of staphylococcusaureus.【Methods】1. We designed and synthesized a76-base ssDNA containing40bases of randomsequence flanked by defined sites in vitro. Staphylococcus aureus enterotoxin A(SEA) was used as a target in the SELEX. The ssDNA aptamers specificallybinding SEA were obtained from the library. Then we used aptamer labeledfluorescence direct assay to identify the binding capabilities of aptamers withinevery one round to SEA.2. PCR products of enriched library were amplified, pured, cloned and sequenced.Chromas and DNAman softwares were employed to analyze the primarystructure and prediction secondary structure of the aptamers.3. The affinity and specificity of aptamer was identified by enzyme-linked aptamerdirect assay and dot hybridization assay, according to Digoxigenin-anti-digoxigenin-AP system and biotin-streptavidin-AP system, separately. Thedissociation constant (Kd) was obtained through softwares that analysised thebinding capabilitie of aptamers in the different concentration.4. After serious of concentration aptamer binding SEA, the mitogen activity of SEAwas determined monitor the stimulation index (SI) of PBMC with MTT. 【Results】1. The results showed that the binding capabilities between ssDNA and SEA hadbeen more strength following the selection increasing. The BR of the ninth roundwas up to48.3%，8fold compared with the first round (5.8%). The bindingssDNA aptamers were not increasing when the binding sites were full.2. We cloned and sequenced the PCR products of9th round selection.17sequenceswere classified4groups which were S3, S12, S6, S23. The prediction secondarystructure of the aptamers mainly consisted hairpin and stem-loop.3. We used ELADA and DHAto determine the binding capabilities and specificitiesof aptamers. It had been shown that aptamers bound only to SEA but not to otherprotein. The dissociation constant (Kd) of aptamer S3（36.57nM）was lesser thanS6(75.62). S3is better than S6.4. Proliferation of human PBMC was inhibited by aptamer when the concentrationabove250nmol/L. The stimulation index（SI）was cut down to1.40. It is said thataptamer can block the superantigen activity of SEA.【Conclusions】we have successfully gotten the aptamer S3with high affinity and specificityagainst SEA by SELEX，which also inhibitsthe superantigen activity of SEA. ThessDNA aptamer specifically binding SEA can provide a foundation for investigationand therapy infection of caused by SEA, so it has good prospect in use.