In Vitro Selection Of Aptamers To Staphylococcal Enterotoxin B(SEB) By SELEX And Application

【Objective】To obtained single strand DNA(ssDNA) aptamers specifically binding to Staphylococcal enterotoxin B (SEB) which were selected from a liborary contained 60 random sequences at totall length 96 in vitro by a method of systematic evolution of ligands by exponential enrichment (SELEX).Basing on a high affinity aptamers found,a Enzyme-linked aptamer immunopsorbent assay and a colloidal gold labelled aptamers sandiwish dot filtration assay were developed to detect SEB. We invested the inhibitory effects of aptamers on the mitogen activity of SEB.【Methods】1. A 96-base ssDNA containing 60 bases of random sequence flangked by defined sites library was designed and synthesized in vitro. It was using Staphylococcal enterotoxin B (SEB) as target in the SELEX. The ssDNA aptamers specifically binding SEB were obtained through selection. The binding capabilities of aptamer within every one round to SEB were identified by aptamer labeled fluorescence direct assay.2. We detected the absorbance of Enzyme-linked oligonucleotide to determine the binding capabilities of aptamers between SEB and other proteins according to Digoxigenin -anti-digoxigenin-AP system. The dissociation constant (Kd) was obtained through softwares that analysised the binding capabilitie of aptamers in the different concentration. We also established an oligonucleotide labelled colloidal gold assay to detect the specificity of aptamers ability .3. PCR products of the 11 rounds of selection were cloned and sequenced. Relative softwares were employed to analyze the primary structure and prediction secondary structure of the aptamers. 4. We finished a Enzyme-linked aptamer immunopsorbent assay (ELASA) which use an anti-SEB antibody coated microwell plates for capture and a Enzyme-labeled conjugate of aptamers for reportor to quantitate SEB.Then we experimented the preparation of the colloidal gold labelled with aptamers that used as probes to established a dot immuno filtration assay. On the basis of it, we developed a double aptamers dot filtration assay (DADFA) which coated aptamers on nitrocellulose (NC)membrane for capturing SEB to detect sensiticity rapidly.5. In order to observe the inhibitory effects of aptamers on the function of SEB. After serious of concentration aptamer binding SEB, mitogen activity of SEB was determined stimulation index (SI) with MTT .And the IFN-γproduction levels by PBMC was detected with ELISA.【Results】1. The results showed that a significant signal of binding capabilities increased over background following the selection. After 13 rounds of selection, the binding ability of the aptamers was from 1.1% to 39.8%, 36 times more than first round. The binding ssDNA aptamers were not increasing when the binding sites were full.2. Basing on Enzyme-linked oligonucleotid, we detected the absorbatnce to determine the binding capabilities of aptamers between SEB and other proteins. It had been shown that absorbance to SEB reach 1.02,more than SPA (0.35),BSA (0.32) and the bank (0.28) .The dissociation constant (Kd) of aptamer was 21.9 nm which obtained through relative softwares. The result of aptamers specificity from oligonucleotide labelled colloidal gold assay was consistent with ELADA.3. After cloning and sequencing PCR products of the 11 rounds of selection ,we found two cloned of twenty five sequences were mostly enriched.Their homology were above 95%.We applied their multipule sequence aligments by Chromas and DNAman to classified 7 families. DINAMelt sever was used to predicted their secondary structures which mainly consisted of stem-loop,G-tetramer.4. We applied a Enzyme-linked aptamer immunopsorbent assay (ELASA) for determining the concentration of SEB. The curve provided its linear sensitivity detection concentration ranged from 0.1ug/ml to 20ug/ml. We applied a double aptamers dot filtration assay (DADFA) to detect sensitivity of aptamers. The color of the positive dots (red dots)were so bright because of the aptamers labelled gold only specially binding to SEB, but not to SPA,BSA,normal serum or bank.5. Proliferation of human PBMC was inhibited by aptamer when the concentration above 200nmol/ml. The stimulation index (SI) was cut down to 1.03.And the levels of IFN-γsecreted by SEB stimulated PBMC were significantly decreased to 25ng/ml after aptamers stimulation. There were significant differences in statstics.The mitogen activity of human PBMC stimulated by SEB can be blocked by aptamers.【Conclusions】We have successfully selected a suit of aptamers with high affinity and specificity against SEB by SELEX technology. We have successfully established a Enzyme-linked aptamer immunosorbent assay and a colloidal gold labelled aptamers dot filtration assay to detect SEB.The result showed there were good sensitivity and specificity.The cell research found that mitogen activity of human PBMC stimulated by SEB can be blocked by aptamers. The ssDNA aptamers specifically binding SEB had been selected by this method, providing a foundation for investigation and therapy infection of caused by SEB.