CAJ | 학술논문

目的：在核酸适配体与靶蛋白特异结合的凝胶电泳迁移实验（EMSA）测定方法中，探索使用非同位素标记取代同位素标记。方法：取一定量的生物素标记的核酸适配体与靶蛋白（或无关蛋白）于室温孵育，聚丙烯酰胺非变性凝胶电泳后将核酸适配体湿转至带正电荷的尼龙膜上，紫外交联，经蛋白酶K消化后，用化学发光法检测。结果：与未使用蛋白酶K消化组相比，经蛋白酶K消化后，可明显呈现核酸适配体与靶蛋白特异结合后产生的阻滞条带，其灵敏度不次于同位素标记的检测方法。结论：经蛋白酶K消化步骤优化的生物素标记的EMSA方法，可以替代放射性同位素标记的EMSA方法，用于核酸适配体与靶蛋白结合情况的鉴定。
목적：재핵산괄배체여파단백특이결합적응효전영천이실험（EMSA）측정방법중，탐색사용비동위소표기취대동위소표기。방법：취일정량적생물소표기적핵산괄배체여파단백（혹무관단백）우실온부육，취병희선알비변성응효전영후장핵산괄배체습전지대정전하적니룡막상，자외교련，경단백매K소화후，용화학발광법검측。결과：여미사용단백매K소화조상비，경단백매K소화후，가명현정현핵산괄배체여파단백특이결합후산생적조체조대，기령민도불차우동위소표기적검측방법。결론：경단백매K소화보취우화적생물소표기적EMSA방법，가이체대방사성동위소표기적EMSA방법，용우핵산괄배체여파단백결합정황적감정。
Objective: Using non-isotope electrophoretic mobility shift assay(EMSA) to replace isotope-labeled EMSA method for detecting aptamer specifically binding to its target protein. Methods: Biotin-labeled ssDNA ap?tamer incubated with the target protein(or unrelated protein) at room temperature, the ssDNA-protein complex were run onto a non-denaturing polyacrylamide gel electrophoresis and then transfered to nylon membrane. After UV cross-linking, proteinase K digestion, the shift bands were observed by chemiluminescence method. Results:After proteinase K digestion, the shift bands of ssDNA aptamer to its target protein could be clearly observed and the sensitivity is similar to the isotope-labeled EMSA method. Conclusion: The biotin-labeled EMSA method with proteinase K digestion could be used for the detection of binding of ssDNA aptamer to its target.