Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Baqiyatallah Research Center for Gastroenterology and Liver Disease, Baqiyatallah University of Medical Sciences, Tehran, Iran.
J Biotechnol. 2018 Nov 20;286:45-55. doi: 10.1016/j.jbiotec.2018.09.006. Epub 2018 Sep 18.
Aptamers or chemical antibodies are oligonucleotides (DNA or RNA) that are able to bind to various targets with high specificity and affinity such as toxins which are isolated by an in vitro method known as SELEX. To date, there are many SELEX procedures for the isolation of novel aptamers against proteins. However not all modified SELEX are suitable for similar protein based on sequence homology such as staphylococcal enterotoxins. Staphylococcal enterotoxin type A (SEA) is the most prevalent toxin involved in staphylococcal food poisoning (SFP) worldwide. SEA is homologous to Staphylococcal enterotoxin type D (SED) and Staphylococcal enterotoxin type E (SEE) about 50% and 83%, respectively. Here, we have developed Staggered Target SELEX (ST-SELEX) as a novel designed SELEX procedure to acquire specific non-cross-reactive aptamers against SEA as a model protein.
In this study, isolated ssDNA aptamers by ST-SELEX were used for detection of SEA via apta-Real time PCR (apta-qPCR). After in silico analysis of SEA protein with SEE and finding the specific region on the surface of protein, ST-SELEX was carried out in two steps (classical SELEX and Second SELEX). Finally, after isolating high specific aptamers, the apta-qPCR was used for the detection of the SEA. In this technique, poly-clonal antibody against SEA was immobilized on protein G sepharose beads (Ab-PGs). Then, the SEA protein was captured by poly clonal antibody as the target that immobilized on sepharose beads. The isolated aptamers were bound on the surface of SEA protein that captured by Ab-PGs. Finally, the heat-released aptamers were amplified by qPCR.
Our investigation showed that the aptamers were generated in vitro by a ten-round selection process based on ST-SELEX procedure with dissociation constant (K) value 7.44± 0.6 nM and limit of detection (LOD) of 146.67 fM.
The advantage of ST-SELEX compared to other SELEX methods was to select a specific non cross-reactive aptamer against two or more proteins with high sequence homology. These aptamers can be used in sensitive detection methods such as apta-qPCR.
适体或化学抗体是能够与各种靶标高度特异性和亲和力结合的寡核苷酸（DNA 或 RNA），例如毒素，这些毒素是通过称为 SELEX 的体外方法分离的。迄今为止，已经有许多用于分离新型针对蛋白质的适体的 SELEX 程序。然而，并非所有经过修饰的 SELEX 都适用于基于序列同源性的类似蛋白质，例如葡萄球菌肠毒素。葡萄球菌肠毒素 A（SEA）是全球与葡萄球菌食物中毒（SFP）相关的最常见毒素。SEA 与葡萄球菌肠毒素 D（SED）和葡萄球菌肠毒素 E（SEE）的同源性分别约为 50%和 83%。在这里，我们开发了交错靶标 SELEX（ST-SELEX）作为一种新设计的 SELEX 程序，以获得针对 SEA 作为模型蛋白的特异性非交叉反应适体。
在这项研究中，通过 ST-SELEX 分离的 ssDNA 适体用于通过适体实时 PCR（apta-qPCR）检测 SEA。在用 SEE 对 SEA 蛋白进行计算机分析后，发现蛋白表面的特定区域，然后分两步进行 ST-SELEX（经典 SELEX 和第二次 SELEX）。最后，分离出高特异性适体后，使用 apta-qPCR 检测 SEA。在该技术中，针对 SEA 的多克隆抗体固定在蛋白 G 琼脂糖珠上（Ab-PGs）。然后，SEA 蛋白作为固定在琼脂糖珠上的靶标被多克隆抗体捕获。分离的适体结合在 Ab-PGs 捕获的 SEA 蛋白表面。最后，通过 qPCR 扩增释放的适体。
我们的研究表明，基于 ST-SELEX 程序的十轮选择过程在体外产生了适体，解离常数（K）值为 7.44±0.6 nM，检测限（LOD）为 146.67 fM。
与其他 SELEX 方法相比，ST-SELEX 的优势在于能够针对具有高度序列同源性的两种或更多种蛋白质选择特异性非交叉反应的适体。这些适体可用于敏感的检测方法，如 apta-qPCR。
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