SELEX Screening And Molecular Simulation Of Staphylococcal Enterotoxin A’s Aptamers

Staphylococcal enterotoxin A (SEA) is one of the most frequently toxin protein in food, and there are many detection methods, for example biology experiment, equipment technology, and immunological methods. The immunology method is a spreaded because of its easy, rapid, sensitive and specify characters. But the antibody is significantly influenced by environment, which lead to the false positive or negative results. SELEX (Systematic evolution of ligands by exponential enrichment) is a new method, having more application space in foodsafety field, due to its more stable character than immunology method.To optimize the SELEX method, BSA (bovine serum albumin) was used as a representive molecular. Five aptamers for the detection of SEA were selected with the improved SELEX and simulated with Pymol and Molegro Vitual Docker software, and the function of aptamers was initially validated in this paper. The results were listed as follows:1. Optimization of the classic SELEXThe original libraries from nearly twenty study teams were analyzed by Primer 5.0 from the aspects of anneal temperature, GC content, hairpin, dimmer, false priming, cross dimmer and most stable hairpin. The library with sequence:5’-ACC GAC CGT GCT GGACTC T-(N)40-AGT ATG AGC GAG CGT TGC G-3’was selected.In this research, separation methods were optimized from two aspects using BSA as target molecular. At first, the classic solid-SELEX and liquid-SELEX were compared, from which the result that solid-SELEX was more suitable to high weiht moleculors was obtained. On the other hand, different separation materals including mixed cellulose membrane, acetylcellulose membrane, nitrocellulose membrane and nylon membrane were selected by detecting the non-specific products after the same cycles.The results indicated that the adsorption rate of KOH treated mixed cellulose membrane was decreased from 26.01% to 2.95% than conventional treated nitrocellulose membrane. The efficiency was significantly improved.During the amplification process, the orthogonal experiment of symmetric PCR and a symmetric PCR. The result showed that:the annealing temperature and heat cycles in symmetric PCR was 68℃and 10 cycles and the proportion of two primers and heat cycles in asymmetric PCR was 40:1 and 25 cycles, respectively.During the process of separation of sub-library, the result revealed that the method of repeated freezing and thawing had a higher recovery rate.After the optimization of four key processes above, the optimized SELEX significantly improved the efficency. The result revealed that:The unspecific amplification appeared in the 10 cycles with classic SELEX, but it appeared in 15 cycles. That significantly improved the efficency.2. Screening of the SEA’s aptamerAccording to the optimized SELEX, the SEA’s aptamers were obtained. Separation of single aptamer with PCR-SSCP:60 clonies were chosed randomly to analyze with PCR-SSCP, and sent to sequence. And the result showed that:only five right sequence obtained. Most of these were mix base.3. Molocular simulation of aptamersMoleculor simulation of 3-D structures:The 3-D structures of SEA,1-4,1-8,1-13, 2-4,2-8 were obtained with Pymol software. Moleculor docking:The moleculor docking of SEA were carried with Molegro Vitual Docker software software, and only the 1-4, 1-13 and 2-4 aptamers could dock with SEA.